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V(D)J recombination coding junction formation without DNA homology: processing of coding termini.

Coding junction formation in V(D)J recombination generates diversity in the antigen recognition structures of immunoglobulin and T-cell receptor molecules by combining processes of deletion of terminal coding sequences and addition of nucleotides prior to joining. We have examined the role of coding end DNA composition in junction formation with plasmid substrates containing defined homopolymers flanking the recombination signal sequence elements. We found that coding junctions formed efficiently with or without terminal DNA homology. The extent of junctional deletion was conserved independent of coding ends with increased, partial, or no DNA homology. Interestingly, G/C homopolymer coding ends showed reduced deletion regardless of DNA homology. Therefore, DNA homology cannot be the primary determinant that stabilizes coding end structures for processing and joining.

Animals↗

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA.

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.

Adenosine Triphosphate↗

Poly(amino acid) bioadhesives for tissue repair.

We report here on poly(amino acid)-based bioadhesives with potential to bond to soft tissues. The systems investigated included homopolymer poly(amino acids), mixtures of poly(amino acids) and amino acids, and blends of different poly(amino acids). Adhesive performance was tested in tension on glass surfaces, chondroitin sulfate surfaces, as well as bovine cartilage surfaces. The amino acid structural units contained acidic, basic, or polar side chains and were found to adhere reasonably well to the surfaces of glass and chondroitin sulfate. The formation of polymer-monomer complexes with the addition of a basic monomer such as L-lysine to negatively charged polymers such as poly(L-aspartic acid) and poly(L-glutamic acid) was found to result in greater adhesive strength relative to homopolymers. Further improvement in adhesion was observed in blends of poly(L-lysine) with polar poly(amino acids) such as poly(L-asparagine). Adhesion on wet cartilage surfaces was the weakest measured but a priming approach designed to form electrostatic or hydrogen bonds appears promising. We believe the strength of the adhesives studied here is based on the ability of their constituent polymers and monomers to form molecular entanglements and crosslinks for load transfer. We also believe hydrogen bonds and electrostatic forces play a role in the adhesion of the systems to the substrates probed in this work.

Adhesiveness↗

Polyimide-polyethylene glycol block copolymers: synthesis, characterization, and initial evaluation as a biomaterial.

Block copolyimides with varying amounts of polyethylene glycol (PEG) were synthesized and characterized by copolymerization of diaminodiphenyl ether (DDE), amino terminated PEG, and benzophenone tetracarboxylic acid dianhydride (BTDA). Strong materials were obtained, with enhanced flexibility as compared to the parent DDE-BTDA polyimide homopolymer. Incorporation of PEG led to an increase in water absorption by these copolymers, and hydrophilicity was increased as reflected by a decrease in air-water-polymer contact angle. These materials supported less cell adhesion in vitro than the parent polyimide homopolymer. Short term in vivo evaluation of these copolymers showed reduced fibrous encapsulation than was observed in the absence of PEG.

Cell Adhesion↗

Novel PDLLA/PEG copolymer micelles as drug carriers.

In this study we attempted to develop a novel drug delivery system in the form of polymeric micelles. In order to obtain polymer chains with micelle forming abilities, we have proposed to produce copolymers by transesterification of poly(DL-lactic acid)(PDLLA)(as the hydrophobic segment) and polyethylene glycol (PEG) (as the hydrophilic segment). We first produced homopolymers of PDLLA with low molecular weights by condensation polymerization of DL-lactic acid. The PDLLA homopolymer with an average molecular weight of 1866 +/- 100 was then transesterified with PEG with a molecular weight range of 3300-4000. By changing the ratio of PEG to PDLLA, we were able to produce copolymers with different chain structures, and therefore, with different micelle forming abilities. FTIR, 1H-NMR, DSC, and GPC studies were performed to describe the structures of these PDLLA/PEG copolymers. Micelles of these copolymers were characterized by light scattering. We selected a model drug, i.e. adriamycin, and obtained the drug loadings to the PDLLA/PEG copolymer micelles. The maximum drug loading was about 12 mg g-1. We found that these micelles were degraded in phosphate buffer (pH 7.4 and temperature 37 degrees C) in about 5-6 weeks. We also investigated the release of adriamycin from these PDLLA/PEG micelles, and concluded that the drug release from these micelles was mainly 'degradation controlled'.

Biodegradation, Environmental↗

Cell interactions with perfluoropolyether-based network copolymers.

We have investigated the potential of several polymers based on perfluoropolyether (PFPE) macromonomers for use in biomaterial applications. Polymer networks were synthesised from the PFPE macromonomers of increasing chain length and the adhesion and proliferation of corneal, vascular and bone cells was evaluated on these polymers. The polymer surfaces were quite hydrophobic, having sessile air-water contact angles ranging between 96 and 125 degrees. However, these polymers supported the attachment and growth of bovine corneal epithelial and endothelial cells and fibroblasts at 60-100% of the rate of cell growth on the culture substratum, TCPS. Furthermore, the PFPE polymers supported the attachment and growth of vascular endothelial cells (from human umbilical artery) and human bone-derived cells over a 7 day period at an equal level to TCPS. The relationship between the macromonomer chain length (n = 1 to 4) and the ability of the resulting PFPE homopolymer to support the overgrowth of corneal epithelial tissue was also evaluated. The PFPE-containing polymers supported corneal epithelial tissue overgrowth, with the most effective having a performance equivalent to that of TCPS. In addition to these homopolymers, copolymers comprising of PFPE and N,N-dimethylaminoethyl methacrylate (DMAEMA) were also synthesised. Surprisingly, the addition of DMAEMA to the PFPE polymer network lead to a reduction in the growth and attachment of corneal epithelial cells and fibroblasts. These results indicate that PFPE-based materials show a potential for use in the development of biomaterials in the ocular, vascular and orthopaedic areas.

Animals↗

Lupus-derived human monoclonal IgM anti-DNA antibody displays monospecificity, high affinity and private idiotype specificity.

A human monoclonal IgM k anti-DNA antibody, designated 2F7, was prepared by somatic hybridization of peripheral blood lymphocytes from a lupus patient with a human-mouse heterohybridoma cell line, K6H6/B5. 2F7 was tested for its antigen binding and idiotypic specificity by direct binding and inhibition enzyme-linked immunosorbent assays. 2F7 had a high binding activity to single-stranded DNA (ssDNA) but not to double-stranded DNA. It cross-reacted with single-stranded homopolymers with pyrimidine bases and double-stranded polynucleotides containing those homopolymers, suggesting that 2F7 recognizes a conformational determinant made up of both deoxyribose-phosphate backbone and specific nucleotide base. 2F7 did not cross-react with eight structurally unrelated self-antigens. Dissociation constant (Kd) of 2F7 for sonicated ssDNA was approximately 4.5 x 10(-9) M, indicating its relatively high affinity. Idiotypic characterization with rabbit anti-idiotype raised against 2F7 suggested that 2F7 expressed an idiotype at or near its antigen-binding sites that was not detected in sera from 20 unrelated lupus patients, 10 lupus family members and 10 normal individuals. These results suggest that certain IgM class anti-DNA antibodies in human systemic lupus erythematosus may arise by antigen stimulation and not simply by polyclonal B-cell activation.

Animals↗

Isolation and properties of vitellogenic ferritin from snails.

The iron storage protein ferritin is the principal yolk protein in oocytes of the snails Planorbarius corneus L. and Lymnaea stagnalis L. This report gives an account of the isolation procedure and of some properties of snail ferritins. The isolation procedure includes a heat-denaturation step, gel filtration on Sepharose 6B and two ultracentrifugation steps followed by electrophoresis. Ferritins from both snails are highly reminiscent of vertebrate and plant ferritins in terms of heat stability, absorption spectrum and ultrastructure, and both share common antigen determinants with horse spleen ferritin. In different electrophoresis systems snail ferritins display considerable heterogeneity and microheterogeneity. Electrophoresis in the presence of sodium dodecyl sulphate (SDS) yields two major polypeptides with molecular weights of 19 000 and 24 000, which are interpreted to be authentic subunits of the ferritin molecule. Different organs and tissues of the snails differ in subunit composition. Midgut gland ferritin consists predominantly of the 19 000 Mr polypeptide, while in embryos only the 24 000 Mr band was found. No carbohydrates or lipids could be detected by staining acrylamide gels. Results from SDS/acrylamide electrophoresis, electrophoresis under non-denaturing conditions on gradient gels and from isoelectric focusing indicate that the ferritins of both snails are composed of at least two different types of ferritin that are tissue-specific. One ferritin is typical of somatic tissue (midgut gland) and is most probably a homopolymer of the 19 000 Mr subunit. The other ferritin is typical of oocytes, but since it is an exogenous protein it is also encountered in the midgut gland (the presumed site of yolk synthesis) and the haemolymph. Vitellogenic ferritin is either a homopolymer of the 24 000 Mr subunit or is predominantly composed of it. So far, there is no evidence for a precursor-product relationship between the two subunits.

Animals↗

Effect of the strandedness of cofactor DNA on the activities of DNA-dependent ATPases B and C3 from FM3A cells.

The requirements of cofactor DNA for DNA-dependent ATPases B and C3 were analyzed in detail. ATPase B and C3 required the presence of a polynucleotide for their activities. Among the DNAs tested, ATPase B showed a preference for poly(dT) as its cofactor. The other deoxyhomopolymers, except poly(dG) and heat-denatured DNA also were effective. The alternating polydeoxyribonucleotide, poly[d(A-T)] had an efficiency 23% that of heat-denatured DNA. Unlike ATPase B, ATPase C3 showed almost no activity with deoxyhomopolymers. The most effective cofactor for ATPase C3 so far tested is poly[d(A-T)]. Relatively high activity was obtained with heat-denatured DNA. The high activity of ATPase B with poly(dT) was reduced by the addition of poly(dA). The addition of noncomplementary homopolymers did not affect enzyme activity. ATPase C3 activity in the presence of 10 microM poly(dT) increased gradually with concentrations of poly(dA) up to 20 microM, after which it decreased. Almost no increase in activity was observed when noncomplementary homopolymers were added. The relatively high activity of ATPase C3 with heat-denatured DNA was suggested by its high sensitivity to ethidium bromide to be due to the double-stranded region in the heat-denatured DNA formed by self-annealing.

Adenosine Triphosphatases↗

Viscoelastic study of the mechanical unfolding of a protein by AFM.

We have applied a dynamic force modulation technique to the mechanical unfolding of a homopolymer of immunoglobulin (Ig) domains from titin, (C47S C63S I27)5, [(I27)5] to determine the viscoelastic response of single protein molecules as a function of extension. Both the stiffness and the friction of the homopolymer system show a sudden decrease when a protein domain unfolds. The decrease in measured friction suggests that the system is dominated by the internal friction of the (I27)5 molecule and not solvent friction. In the stiffness-extension spectrum we detected an abrupt feature before each unfolding event, the amplitude of which decreased with each consecutive unfolding event. We propose that these features are a clear indication of the formation of the known unfolding intermediate of I27, which has been observed previously in constant velocity unfolding experiments. This simple force modulation AFM technique promises to be a very useful addition to constant velocity experiments providing detailed viscoelastic characterization of single molecules under extension.

Animals↗

Polymers for biodegradable medical devices. IX: Microencapsulation studies; effects of polymer composition and process parameters on poly-hydroxybutyrate-hydroxyvalerate microcapsule morphology.

Reservoir-type microcapsules were prepared using a double emulsion solvent evaporation technique from a series of nine different poly-beta-hydroxybutyrate (PHB)-based polymers in which both molecular weight and hydroxyvalerate content were varied. Particles prepared from a low molecular weight (MW 43,000) homopolymer had a shrivelled appearance, but were not porous. When the molecular weight of the fabricating homopolymer was increased to 540,000, however, these features disappeared and non-wrinkled particles with microporous surfaces were observed. Microcapsules prepared from a high molecular weight copolymer of PHB with 10.8 per cent hydroxyvalerate (HV) had a similar appearance, but particles prepared from a high molecular weight 20.1 per cent HV copolymer had much smoother and less porous surfaces. Lowering the molecular weight of the copolymer had the effect of producing particles that were generally distorted in shape and had highly irregular, macroporous, surface morphologies. Increasing the double emulsion temperature to 40 degrees C at 2 min after phase combination typically generated smoother and/or less porous particles and improved the batch yield. The numerous factors potentially responsible for the development of morphological characteristics of the products are discussed in the light of these observations.

Biodegradation, Environmental↗

Determination of the hydroxyapatite-nucleating region of bone sialoprotein.

Bone sialoprotein (BSP) was shown to be a potent nucleator of hydroxyapatite (HA) in a steady-state agarose gel system (Hunter and Goldberg, 1993, PNAS 90: 8562). Nucleation of HA was also demonstrated with the homopolymer poly-glutamic acid but not with poly-aspartic acid or osteopontin. Since BSP contains contiguous sequences of glutamic acid, it is reasonable to suggest that the HA-nucleating activity of BSP resides within these regions. Purified porcine BSP was treated with trypsin and digests fractionated by gel filtration. In addition to small peptides (P3-5), two peptides of 38 kDa (P1) and 25 kDA (P2) were recovered, and after characterization assigned to the regions within BSP encompassing residues 133-272 (P1) and 42-125 (P2). Each of these peptides contained one of the two glutamic acid-rich regions of porcine BSP. In the steady-state agarose gel system, BSP, P1 and P2 induced HA formation, whereas the pooled small BSP-derived peptides (P3-5) did not. Analysis by circular dichroism spectroscopy revealed that the homopolymer poly-L-glutamic acid assumes a helical structure, while poly-L-aspartic acid does not. These findings suggest that the nucleating activity does not require intact molecules, that the nucleation of HA and BSP appears to require glutamic acid-rich sequences in a helical conformation and that there are two domains in porcine BSP that are each capable of nucleating HA.

Animals↗

Enzymes of nucleotide metabolism: the significance of subunit size and polymer size for biological function and regulatory properties.

The 72 enzymes in nucleotide metabolism, from all sources, have a distribution of subunit sizes similar to those from other surveys: an average subunit Mr of 47,900, and a median size of 33,300. The same enzyme, from whatever source, usually has the same subunit size (there are exceptions); enzymes having a similar activity (e.g., kinases, deaminases) usually have a similar subunit size. Most simple enzymes in all EC classes (except class 6, ligases/synthetases) have subunit sizes of less than 30,000. Since structural domains defined in proteins tend to be in the Mr range of 5,000 to 30,000, it may be that most simple enzymes are formed as single domains. Multifunctional proteins and ligases have subunits generally much larger than Mr 40,000. Analyses of several well-characterized ligases suggest that they also have two or more distinct catalytic sites, and that ligases therefore are also multifunctional proteins, containing two or more domains. Cooperative kinetics and evidence for allosteric regulation are much more frequently associated with larger enzymes: such complex functions are associated with only 19% of enzymes having a subunit Mr less than or equal to 29,000, and with 86% of all enzymes having a subunit Mr greater than 50,000. In general, larger enzymes have more functions. Only 20% of these enzymes appear to be monomers; the rest are homopolymers and rarely are they heteropolymers. Evidence for the reversible dissociation of homopolymers has been found for 15% of the enzymes. Such changes in quaternary structure are usually mediated by appropriate physiological effectors, and this may serve as a mechanism for their regulation between active and less active forms. There is considerable structural organization of the various pathways: 19 enzymes are found in various multifunctional proteins, and 13 enzymes are found in different types of multienzyme complexes.

Enzymes↗

The effect of polymer coating biomaterials individually and incombination on the behavior of transformed RAW macrophage cells.

The use of homopolymers as coatings for biomaterials has received much attention in the last decade. However, the modifications and alterations induced by using such materials towards inflammatory cells have not been fully investigated. The specific objectives of this study were to investigate the role of hetero and homopolymers of amino acids on cell proliferation, and to determine the biochemical behavior associated with the incubation of RAW cells with various polymers. The RAW macrophage cell line was obtained from the American Type Culture Collection (Rockville, MD) and maintained in sterile media (RPMI) supplemented with 10% fetal bovine serum and 1% antibiotics and antimycotics. The cells were plated at a density of 1 x 10(6) cells/ml onto 24 well plates. The plates were divided into four groups of six wells per group per phase (24, 48, and 72 hours). Cells in the first group were treated with RGD, cells in group II were treated with poly-L-lysine, group III cells were treated with RGD + poly-L-lysine, and cells in group IV were treated with media alone, and served as controls. Cell number, as well as, protein, MDA, lactate dehydrogenase (LDH), and cytochrome C (cyto C) were determined at the end of 24, 48 and 72 hours. Data obtained from this investigation revealed that: (I) there were no significant difference in total cell counts between all experimental groups and control at the end of the 48 hour phase. However, at 24 hours there were fewer cells in the poly-L-lysine treated wells in comparison to control group (p < 0.05), (II) RGD and poly-L-lysine treatments did not cause changes in MDA or protein concentrations for the entire duration of the experiment, and (III) RGD treatment for 48 and 72 hours did not cause a reduction in the LDH activity compared to control and poly-L-lysine treated groups. Data obtained from this investigation could provide more insight regarding the design and development for safe and biocompatible orthopedic, dental and drug delivery devices.

Biocompatible Materials↗

Poly(8-bromoadenylic acid): synthesis and characterization of an all-syn polynucleotide.

Poly(8-bromoadenylic acid) (poly(8-BrA)) has been synthesized by polymerization of 8-BrADP with polynucleotide phosphorylase in the presence of oligonucleotide primers. In the absence of oligonucleotides, significant (i.e. more than 1%) polymerization does not occur. Oligo(I) primer was removed selectively from the polymer with ribonuclease T1 to yield the homopolymer, poly(8-BrA). End group analysis, based on quantitative infrared measurement of the (Ip)3I-primed polymer, indicates an average degree of polymerization of about 70 residues. The primed polymers and the homopolymer appear to have similar helical structures, probably double-stranded with mutual hydrogen bonding interaction of BrA residues. Preliminary NMR observations of poly(8-BrA) with a tetrainosinic acid primer at the 5' ends of the polymer chain ((Ip)3I-(8-BrA)n) are consistent with the existence of a rigid helical structure below the melting range of the primed polymer. Above the melting range (81 degrees) the H1' coupling constants of (Ip)3I-(8-BrA)n and of polyadenylic acid (poly(A)) suggest a significantly higher population of C3' endo conformation of ribose residues in the primed polymer than in poly(A) at 81 degrees.

Adenosine Monophosphate↗

Relative rates for plasma homo- and copolymerizations of olefins in a homologous series of fluorinated ethylenes.

It is well known that the rate of plasma polymerization, or deposition rate, of a given monomer depends on various plasma process parameters, e.g., monomer flow rate, pressure, power, frequency (DC, rf or microwave), location of the substrate in the reactor, reactor geometry or configuration, and temperature. In contrast, little work has been done to relate deposition rates to monomer structures for a homologous series of monomers where the rates are obtained under identical plasma process parameters. For the particular series of fluorinated ethylenes (C2HxF4-x; x = 0-4), deposition rates were reported for ethylene (ET), vinyl fluoride, vinylidene fluoride and tetrafluoroethylene (TFE), but for plasma polymerizations carried out under different discharge conditions, e.g., pressure, current density, and electrode temperature. Apparently, relative deposition rates were reported for only two members of that series (ET, x = 4, and TFE, x = 0) for which the plasma polymerizations were conducted under identical conditions. We now present relative deposition rates for both homopolymerizations and copolymerizations of the entire series of fluorinated ethylenes (x = 0-4). Our interest in such rates stems from prior work on the plasma copolymerization of ET and TFE in which it was found that the deposition rates for the plasma copolymers, when plotted versus mol % TFE in the ET/TFE feed stock, followed a concave-downward curve situated above the straight line joining the deposition rates for the plasma homopolymers. This type of plot (observed also for an argon-ET/TFE plasma copolymerization) indicated a positive interaction between ET and TFE such that each monomer apparently "sensitized" the plasma copolymerization of the other. Since the shape of that plot is not altered if mol % TFE is replaced by F/C, the fluorine-to-carbon ratio, this paper aims (1) to show how the relative deposition rates for plasma copolymers drawn from all pairs of monomers in the C2HxF4-x series, as well as the deposition rates for the individual plasma homopolymers, vary with F/C ratios of the monomers or monomer blends, and (2) to see if those rates give rise to a common plot.

Chemistry, Organic↗

[Blood compatibility of block copolymer membranes of poly(benzyl L-glutamate)/poly(ethylene glycol)].

The blood compatibility of block copolymer membranes of poly(benzyl L-glutamate)/poly(ethylene glycol) and the effect of on the blood compatibility of copolymer were evaluated by the clotting time test, the platelet adhesion and deformation test, and the protein adsorption test. The results showed that in terms of blood compatibility, homopolymer was better than glass and silicone, copolymer was better than homopolymer, and the more the PEG in the copolymer, the better the blood compatibility.

Biocompatible Materials↗

Synthesis and characterization of polyanhydride for local BCNU delivery carriers.

p-Carboxyphenoxy propane (CPP) prepolymer consisting of 4 units and sebacic acid (SA) prepolymer consisting of about 10 units were synthesized by reacting CPP and SA in the presence of excess acetic anhydride, respectively. Polyanhydride, poly(CPP-SA) copolymers were copolymerized by a melt polycondensation process with a mixture of CPP and SA prepolymer. Copolymers of average molecular weight up to 110,000 g/mol were achieved. The crystallinity of poly(CPP-SA) copolymers was decreased by the addition of the CPP homopolymer segment to SA homopolymer. Poly(CPP-SA) copolymers gradually degraded for period of 10 days. No large difference of weight loss observed according to molecular weight variation of poly(CPP-SA) copolymers. BCNU release from wafers fabricated by poly(CPP-SA) showed a sustained release pattern with no initial burst and delay of drug release.

Anhydrides↗