Structured drug-eluting bioresorbable films: microstructure and release profile.

Bioresorbable drug-eluting films can be used in many biomedical applications. Examples for such applications include biodegradable medical support devices which combine mechanical support with drug release and antibiotic-eluting film coatings for prevention of bacterial infections associated with orthopedic implants or during gingival healing. In the current study, bioresorbable drug-loaded polymer films are prepared by solution processing. Two film structures are studied: A polymer film with large drug crystals located on its surface (A-type) and a polymer film with small drug particles and crystals distributed within the bulk (B-type). The basic mode of drug dispersion/location in the film (A or B-type) is found to be determined mainly by the process of film formation and depends mainly on the solvent evaporation rate, whereas the drug's hydrophilicity has a minor effect on this structuring process. Most release profiles from A-type films exhibit a burst effect of approximately 30% and a second release stage that occurs at an approximately constant rate and is determined mainly by the polymer weight loss rate. An extremely high burst release is exhibited only by a very hydrophilic drug. The matrix (monolithic) nature of the B-type film enables release profiles that are determined mainly by the host polymer's degradation profile, with a very low burst effect in most of the studied systems. In addition to the drug location/ dispersion in the film, the host polymer and drug type also strongly affect the drug's release profile from the film. It has been demonstrated that appropriate selection of the process parameters and film components (polymer and drug) can yield film structures with desirable drug release behaviors. This can lead to the engineering of new bioresorbable drug-eluting film-based implants for various applications.

Fate of the chemical warfare agent VX in asphalt: a novel approach for the quantitation of VX in organic surfaces.

VX is one of the most toxic chemical warfare agents. Its low volatility and its persistence in the environment raise the issue of long-term exposure risks, either by inhalation or by transdermal penetration. Therefore, a topic of acute interest is the fate of VX in preservative environmental surfaces. In this work, the fate of VX in asphalt pavement, a suspected preservative matrix, was explored, by applying a novel quantitative method for the extraction of trapped VX from "digested" asphalt. It is based on dissolution of asphalt in toluene, precipitation of the heavy components by basic methanol followed by GC-NPD analysis. This method is complementary to methanol extraction of VX from the outer surface of asphalt, and enabled us to explore the total amount of viable VX both on and inside the matrix. Using this method, bis-diisopropylaminoethyl-disulfide [(DES)2], a degradation product of VX, was also assayed. Small chunks of Asphalt were spiked with VX, sealed and analyzed after various aging periods up to 425 days. The level of VX on the outer surface of the asphalt was found to be diminishing with time following a single-exponential decay. The level inside the asphalt increases during the first day, decays steeply to a level of about 5% during the following two weeks, and declines moderately during all the period up to 425 days following a bi-exponential decay. The total recovery of VX from the asphalt declined from almost 100% after 30 minutes to about 2% after 425 days, with a half-life of about 14 days.

Degradation of VX and sulfur mustard by enzymatic haloperoxidation.

Chloroperoxidase (CPO) isolated from Caldariomyces fumago (20 U ml(-1)) together with urea hydrogenperoxide (UPER, 0.5 mM) and sodium chloride as co-substrate (NaCl, 0.5 M) caused rapid breakdown of VX (10 microM) (t((1/2)) = 8 s, 25 C, 50 mM tartarate, pH 2.75). Glucose oxidase (GOX, Aspergillus niger) and glucose were used as an alternative source for H(2)O(2). A mixture of GOX (20 U ml(-1)), glucose (GLU 0.45 M), CPO (20 U ml(-1)) and NaCl (0.5 M) caused a 3.8-fold slower degradation of VX (10 microM) (t((1/2)) = 30 s, 25 C, 50 mM tartarate, pH 2.75). The concentrations of H(2)O(2) and chlorine produced by this enzyme/substrate mixture depended mainly on the GLU concentration. Horseradish peroxidase (HRP) together with UPER (1 mM) and sodium iodide (NaI, 0.05 M) caused progressive degradation of VX that was more than 400-fold slower than with CPO (20 U ml(-1)), UPER (0.5 mM) and NaCl (0.5 M) (t((1/2)) = 55 min, 25 C, pH 8). Skin decontamination of VX by CPO was tested in pig-ear skin in vitro. The chemical agent VX (0.01 M, 100 microl) was degraded by 98% within 3 h of skin diffusion when a mixture of UPER/NaCl/CPO was applied 60 min prior to VX application. A mixture of UPER/NaCl without CPO also caused significant VX degradation (94%) during skin diffusion whereas it did not cause any VX degradation in solution. Degradation of VX in skin, obtained without exogenous CPO, may indicate involvement of endogenous intradermal haloperoxidase-like enzyme. Reagent UPER (1 mM) did not cause any degradation of VX in solution or during its skin diffusion. Furthermore, a mixture of CPO, UPER and NaCl caused rapid degradation of sulfur mustard (HD). Sulfur mustard (50 microM) incubated in the presence of CPO (4 U ml(-1)), UPER (0.05 M) and NaCl (0.5 M) at pH 2.75 and 30 C was oxidized by 97% and 99% within 5 and 10 min, respectively. The oxidation products HD sulfoxide, HD sulfone and HD sulfoxidevinyl were identified by GC/MS in the enzymatic chloroperoxidation mixture.

Ribosomal DNA evidence and disjunctions of western American Portulacaceae.

Phylogenetic analysis of ribosomal DNA internal transcribed spacer sequences from 35 members of western American Portulacaceae plus seven Portulacaceae outgroups generally supports morphologically based interpretations of multiple intercontinental disjunctions. The data neither support nor refute monophyly of the western American group but strongly support a group comprising the western American taxa plus Phemeranthus, the only strictly American genus of the morphology-based eastern American/African group of Portulacaceae, along with the Australian genus Parakeelya. Support is strong for the monophyly of Calandrinia, Montiopsis, Lewisia, Claytonia, and Montia, along with a sister relationship of the last two. The data neither strongly support nor refute the morphologically based diagnosis of Cistanthe, but they strongly support a clade including the North American Cistanthe section Calyptridium and the South American Cistanthe sections Amarantoideae and Philippiamra. The internal transcribed spacer data fail to resolve the phylogenetic relationships among most of the western American lineages, suggesting either rapid radiation or, alternatively, erratic evolution of the internal transcribed spacer. The internal transcribed spacer and morphological evidence together suggest that in this group there have been 8-13 dispersal and colonization events across >2000 km (1 for every 15-26 extant species in this group). The internal transcribed spacer data document complex molecular evolutionary patterns, including strong substitution biases, among-site rate heterogeneity, positional bias for deamination-type substitutions, nonstationarity, and variable rates of insertion/deletion. Our phylogenetic conclusions, however, do not appear to be sensitive to these patterns.

Ligation-mediated PCR for chromatin-structure analysis of interphase and metaphase chromatin.

Chromatin structure is becoming increasingly recognized as important for a full understanding of gene function and cell memory. With regard to cell memory, which involves the transfer of chromatin-encoded epigenetic information from one cell generation to another, the detailed structure of metaphase chromatin is of crucial importance. In this paper we describe methods for the use of dimethyl sulfate, DNase I, and potassium permanganate for in vivo footprinting and chromatin analysis, with special emphasis on studies of metaphase cells. We review the use of ligation-mediated PCR for the analysis of chromatin, including the human phosphoglycerate kinase promoter, and also report initial studies of a matrix attachment region near the human beta-interferon gene.

Deep-level diagnostic value of the rDNA-ITS region.

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.

Conservation patterns in angiosperm rDNA ITS2 sequences.

The two internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA have become commonly exploited sources of informative variation for interspecific-/intergeneric-level phylogenetic analyses among angiosperms and other eukaryotes. We present an alignment in which one-third to one-half of the ITS2 sequence is alignable above the family level in angiosperms and a phenetic analysis showing that ITS2 contains information sufficient to diagnose lineages at several hierarchical levels. Base compositional analysis shows that angiosperm ITS2 is inherently GC-rich, and that the proportion of T is much more variable than that for other bases. We propose a general model of angiosperm ITS2 secondary structure that shows common pairing relationships for most of the conserved sequence tracts. Variations in our secondary structure predictions for sequences from different taxa indicate that compensatory mutation is not limited to paired positions.

Metaphase chromosome analysis by ligation-mediated PCR: heritable chromatin structure and a comparison of active and inactive X chromosomes.

We report that ligation-mediated PCR (LMPCR) can be used for high-resolution study of metaphase chromosomes, and we discuss the role of metaphase chromatin structure in the preservation of differentiated cell states. The X chromosome-linked human PGK1 (phosphoglycerate kinase 1) promoter region was investigated, and euchromatic active X chromosome (Xa) metaphase chromatin was compared with interphase Xa chromatin and to heterochromatic inactive X chromosome (Xi) metaphase and interphase chromatin. We find that (i) good-quality data at single-nucleotide resolution can be obtained by LMPCR analysis of dimethyl sulfate-treated intact metaphase cells; (ii) transcription factors present on the Xa promoter of interphase chromatin are not present on metaphase chromatin, establishing that the transcription complex on the PGK1 promoter must form de novo each cell generation; and (iii) the dimethyl sulfate reactivity pattern of Xa and Xi chromatin at metaphase is very similar to that of naked DNA. These results are discussed in the context of models for heritable chromatin structure and epigenetic mechanisms for cell memory, and they are also relevant to more general aspects of chromatin structure and differences between euchromatin and heterochromatin.

Transient gene expression of foreign genes in preheated protoplasts: stimulation of expression of transfected genes lacking heat shock elements.

Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled, linearized and single-stranded DNA structures that stimulation of transient gene expression in preheated protoplasts was neither dependent on the reporter gene nor on the regulatory elements used. Heat treatment at 42 degrees C also increased expression in protoplasts transfected with a plasmid bearing the tobacco mosaic virus (TMV) translational enhancer, omega. Northern blot analysis revealed that heat treatment of protoplasts before the transfection event greatly increased the amount of the newly synthesized transcripts. Preheating of protoplasts did not affect the transfection efficiency, namely the number of transfected cells in the population, nor the amount of DNA in transfected nuclei, as was inferred from histochemical staining and Southern blot analysis, respectively. The possible mechanism by which heat treatment stimulates transient gene expression of genes lacking obvious heat shock elements is offered. The relevance of the present findings to transient gene expression in plants in general and to viral gene expression in particular is discussed.

Effect of CpG methylation on gene expression in transfected plant protoplasts.

Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M.HpaII (methylates C in CCGG sites) and M.HhaI (methylates GCGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M.SssI, which methylates all CpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all CpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of CpNpG sites in plant cells may, therefore, play a role other than gene silencing.

Externally added DNA molecules support initiation of transcription in isolated nuclei from petunia.

Nuclei isolated from protoplasts transfected with the pUC8CaMVCAT and pDO432 plasmids were able to support, in run off experiments, the synthesis of specific transcripts as was evident from analysis by dot blot hybridization. Also the addition of the above plasmids to nuclei, prepared from non-transfected protoplasts, supported the synthesis of specific transcripts. Dot blot analysis showed that most of the transcripts obtained were complementary to the relevant gene sequences. alpha-Amanitin, at concentrations which are known to block the activity of RNA polymerase II, significantly inhibited the synthesis of specific transcripts by the isolated nuclei. The transcription activity was found to be predominantly associated with the nuclear fraction while the transcription products (RNA molecules) appeared in the supernatant obtained following sedimentation of the nuclei.

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells.

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.

Use of reconstituted Sendai virus envelopes for fusion-mediated microinjection of double-stranded RNA: inhibition of protein synthesis in interferon-treated cells.

Poly(I).poly(C) molecules were trapped with reconstituted Sendai virus envelopes when added to the reconstitution system. A quantitative estimation indicated that about 10% of the added poly(I).poly(C) remained associated with the fusogenic viral envelopes. About 50% of the associated poly(I).poly(C) were found to be RNAase A resistant, enclosed within the viral envelopes. Incubation of loaded viral envelopes with HeLa or L-cells resulted in strong inhibition of protein synthesis, indicating fusion-mediated microinjection of the enclosed poly(I).poly(C). Introduction of poly(I).poly(C) into cultured cells by the use of reconstituted Sendai virus envelopes was as efficient as the introduction of these polynucleotides using the calcium phosphate coprecipitation technique. The inhibition of protein synthesis in L-cells but not in HeLa cells was dependent upon pretreatment with interferon. Incubation of poly(I).poly(C)-loaded viral envelopes with interferon-treated variant cells of the NIH 3T3 line, which possess a very low amount of RNAase L, resulted in only 25% inhibition of protein synthesis, compared to 85% inhibition observed in L-cells.

A new method for reconstitution of highly fusogenic Sendai virus envelopes.

A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.