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Biomedical subjects

L A Chrisey

Publications and source records attributed to L A Chrisey.

14 recordsLinked to original sources
PubMed2001

Surface patterning of polychloromethylstyrene films.

We describe and characterize a simple process for the fabrication of patterned materials on polychloromethylstyrene thin film surfaces under ambient conditions. Patterned deep UV exposure (approximately 60 mJcm(-2), 193 nm) efficiently oxidizes the surface C-Cl bonds of the polymer film, producing an aldehyde species as the major photoproduct. Reductive amination in the presence of ammonium ion and cyanoborohydride reductant selectively converts the aldehyde into an alkylamine, which leads to an amine reactivity template on the film surface. The amines formed are sufficiently reactive to selectively and covalently bind fluorescent dye or electroless Ni metal to the template, which results in negative tone features with micron-scale resolutions (mask limited) in each case. Spectroscopic characterizations of the polymer surface following the photochemical transformation, reductive amination, and grafting steps are presented in support of the process. A key advantage of the method is the use of safe solvents, such as water or simple alcohols, to effect the reductive amination and grafting reactions. This approach mitigates waste disposal and associated environmental concerns, increasing the attractiveness of our method for use with high-throughput track-line processing equipment.

Journal Article↗
PubMed2000

Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription/translation reactions.

We have developed a novel biochemical method to simultaneously amplify and immobilize a target gene onto insoluble particles using PCR. This method employs the covalent attachment of one of two PCR primers to a particle surface either directly during DNA synthesis of the primer or post-DNA synthesis, through the use of chemical crosslinkers. Immobilization of the target gene can be achieved directly during PCR amplification, with one bead-bound primer and one soluble primer. Alternatively, this can be achieved post-PCR, through covalent attachment of a chemically modified primer incorporated into the amplicon to an activated particle. All of the immobilized DNA templates containing appropriate regulatory regions were fully competent for transcription and translation reactions and several could be re-used in serial reactions. The most successful strategy utilized amino-silanized controlled pore glass beads, which were coupled to phosphorylated primers using carbodiimide chemistry. These bead-bound primers were used during PCR to generate attached DNA templates that could be collected and re-used for at least seven sequential transcription reactions without significant loss in efficiency. This method has also been successfully applied to the amplification, transcription and translation of multiple DNA templates using a single, immobilized primer. The combined PCR-based amplification/immobilization method was shown to be more durable than post-PCR chemical immobilization and affords the convenience of performing sequential PCR amplification, transcription and translation reactions in a single tube.

Animals↗
PubMed1996

Covalent attachment of synthetic DNA to self-assembled monolayer films.

The covalent attachment of thiol-modified DNA oligomers; to self-assembled monolayer silane films on fused silica and oxidized silicon substrates is described. A heterobifunctional crosslinking molecule bearing both thiol- and amino-reactive moieties was used to tether a DNA oligomer (modified at its terminus with a thiol group) to an aminosilane film formed on silica surfaces. A variety of aminosilanes, crosslinkers and treatment conditions have been tested to identify optimal conditions for DNA immobilization using this approach. The DNA films which result have been characterized using UV spectroscopy, water contact angle measurement, radiolabeling and hybridization methods.

Cross-Linking Reagents↗
PubMed1996

Fabrication of patterned DNA surfaces.

Two photolithographic methods are described for the formation of patterned single or multiple DNA species on SiO2 substrates. In the first approach, substrates are treated with a photochemically labile organosilane monolayer film. Irradiation of these surfaces with patterned deep UV (193 nm) light results in patterned chemically reactive groups which are then reacted with heterobifunctional crosslinking molecules. Covalent attachment of modified synthetic DNA oligomers to the crosslinker results in stable DNA patterns. Alternatively, a photoresist is spin-coated over a silane film which had been previously modified with the heterobifunctional crosslinker. Upon patterned irradiation and subsequent development, the underlying crosslinker-modified layer is revealed, and is then reacted with a chemically modified DNA. Feature dimensions to 1 micron are observed when a single fluorescent DNA is attached to the surface. By performing sequential exposures, we have successfully immobilized two distinguishable DNA oligomers on a single surface. Synthetic DNA immobilized in this manner retains the ability to hybridize to its complementary strand, suggesting that these approaches may find utility in the development of miniaturized DNA-based biosensors.

Biosensing Techniques↗
PubMed1995

Expression of the Neurospora crassa metallothionein gene in Escherichia coli and its effect on heavy-metal uptake.

The gene coding for the Neurospora crassa metallothionein protein was chemically synthesized and cloned into the fusion expression vectors pMal-c and pMal-p. Cell-fractionation experiments demonstrated the proper localization of the pMal-c and pMal-p- expressed proteins to the cytosol and periplasm of the bacteria respectively. Control bacteria as well as the recombinant bacteria producing the metallothionein protein were incubated with solutions of 109Cd at concentrations of 0.2 microM, 1 microM, and 10 microM. The recombinant bacteria were able to accumulate significantly more 109Cd than control bacteria at all concentrations tested. Cadmium accumulation was rapid and highly selective. Maximum uptake was achieved at a pH of 7.0, with lower accumulation at lower or higher pH values. The pH-dependent uptake of cadmium by the recombinant bacteria was exploited to strip off the bound cadmium from the recombinant bacteria and to regenerate most of the cadmium-binding sites. These observations suggest the potential for using a metallothionein-based biosorbent for certain heavy-metal removal applications.

ATP-Binding Cassette Transporters↗
PubMed1995

Nonsequence-specific inhibition of bacterial luminescence by phosphorothioate oligodeoxyribonucleotides.

To evaluate the effect of synthetic DNA oligomers on regulation of bacterial genes in vivo, we tested 63 oligomers of variable length and chemistry for their ability to selectively suppress light production in the bioluminescent marine organism, Vibrio fischeri. Phosphodiester, phosphorothioate, and mixed backbone oligomers were designed to be lux gene targeted or nontargeted (negative) controls. Although significant suppression of luminescence was observed, most notably with the phosphorothioate oligomers, there was no correlation between inhibitory activity and oligomer sequence. The phosphorothioate oligomer that was most potent for inhibition of luminescence in bacterial culture had no effect on the activity of purified luciferase. Mechanisms other than sequence-specific inhibition of gene expression or direct interaction with luciferase are discussed.

Base Sequence↗
PubMed1994

Direct measurement of the forces between complementary strands of DNA.

Interaction forces between single strands of DNA were measured with the atomic force microscope by a procedure in which DNA oligonucleotides were covalently attached to a spherical probe and surface. Adhesive forces measured between complementary 20-base strands fell into three distinct distributions centered at 1.52, 1.11, and 0.83 nano-newtons, which are associated with the rupture of the interchain interaction between a single pair of molecules involving 20, 16, and 12 base pairs, respectively. When a third long DNA molecule was coupled between complementary surfaces, both intra- and interchain forces were observed. The intrachain interaction resulting from the molecule's elasticity manifested itself as a long-range cohesive force.

Base Composition↗
PubMed1994

Non-sequence-specific antimalarial activity of oligodeoxynucleotides.

The effects of exogenously applied oligodeoxynucleotides on Plasmodium falciparum proliferation was investigated. A fluorescence-activated cell sorter assay was employed to measure parasitemia after administration of either phosphodiester or phosphorothioate oligodeoxynucleotides. We report sequence-independent antimalarial activity preferentially with phosphorothioate congeners with IC50 values in the 1-2 microM range. Phosphorothioate oligodeoxynucleotides which were antisense, sense or nonsense to Plasmodium mRNA, as well as homopolymers (30-mers containing all A or T bases) were equally effective inhibitors of parasitemia. The antimalarial activity was dependent upon oligomer length, concentration, and time of addition to the cultures but was independent of the parasite strain tested. Four P. falciparum strains, including a multi-drug-resistant strain (MDR-K), a drug-sensitive strain (FCR-3), a erythrocyte membrane sialic acid-independent strain (7G8) and a strain isolated from a cerebral malaria patient (CM-87) were equally susceptible to treatment with a phosphorothioate oligomer. Inhibition of red cell invasion is primarily responsible for the observed decrease in proliferation as determined by a study of parasite maturation in the presence of a 30-mer nonsense phosphorothioate oligodeoxynucleotide.

Animals↗
PubMed1993

Internalization of oligodeoxyribonucleotides by Vibrio parahaemolyticus.

The bacterium Vibrio parahaemolyticus was tested for its ability to internalize unmodified as well as modified DNA oligomers without attempting to permeabilize the cells. These experiments were conducted to establish whether it may be feasible to employ antisense oligomers for control of gene expression in Vibrio species without heat-shocking or electroporating the cells. The bacterium was found to bind radiolabeled synthetic oligodeoxyribonucleotides that were added to culture media. Incorporation of a phosphorothioate oligomer into subcellular regions was determined following cellular fractionation. The phosphorothioate was recovered primarily from the periplasm and peptidoglycan layer of the bacterium; however, a significant fraction was recovered from the bacterial cytosol. The extent of uptake depended on both the concentration of oligomer as well as culture medium selected. A maximum of 2.1 x 10(6) oligomers/cell was achieved when a 12-mer phosphorothioate oligomer (10 microM) was added to bacterial cultures in an artificial seawater (Instant Ocean) medium. Several terminally modified oligomers were found to become associated with bacterial cells, albeit less efficiently than the phosphorothioate. None of the oligomers tested was toxic to the bacteria at 0.1 microM, and the phosphorothioate was only marginally toxic at 10 microM. Stability of the oligomers in extracellular and cell-associated fractions was evaluated by PAGE; even after 8 hr of incubation intact phosphorothioate oligomer could be found in both components.

Base Sequence↗
PubMed1991

Uptake of oligodeoxyribonucleotides by lymphoid cells is heterogeneous and inducible.

Oligonucleotide uptake was studied in cultured murine spleen and lymph node cells using internally radiolabeled and fluorescein-5-isothiocyanate (FITC)-labeled oligonucleotides. Lymphoid subpopulations were distinguished by flow cytometry and staining with antibodies to cell-surface molecules. Approximately 5% of fresh lymphoid cells take up substantial amounts of oligonucleotide. The percentage of B cells that take up oligonucleotide increased fivefold if cells were cultured for at least 24 hr prior to incubation with labeled oligonucleotides, and increased 10-fold if cells were precultured for 48 hr. T-cell uptake changed very little in culture. Cultured CD4+ and CD8+ T cells had similar oligonucleotide uptake that was less than one-third of that in cultured B cells, but CD4-CD8- T cells had a higher percentage of cells taking up oligonucleotide than did B cells. T- or B-cell mitogens caused markedly increased oligonucleotide uptake in T or B cells, respectively. Oligonucleotide uptake could be inhibited only partially with competitor DNA. To distinguish between cell membrane-bound and intracellular oligonucleotide, cells were washed in acid glycine buffer (which removes most surface oligonucleotide). This demonstrated that most of the oligonucleotide was intracellular. We conclude that oligonucleotide uptake is quite heterogeneous among cultured cells, and that this uptake is inducible by mitogens. These data may be important for the design and interpretation of in vitro experiments, and for the planning of in vivo therapy with antisense oligonucleotides.

Animals↗
PubMed1990

Inhibition of monoamine oxidases A and B by simple isoquinoline alkaloids: racemic and optically active 1,2,3,4-tetrahydro-, 3,4-dihydro-, and fully aromatic isoquinolines.

A series of 1,2,3,4-tetrahydro-, 3,4-dihydro-, and fully aromatic isoquinolines were tested as substrates and/or inactivators of highly purified human monoamine oxidase A and B (MAO A and B). None were found to be a substrate for either enzyme, but many of these isoquinolines could selectively inhibit either MAO A or B. Stereoselective competitive inhibition of MAO A was found with the R enantiomer of all the stereoisomers tested, including salsolinol (Ki = 31 microM), salsoline (Ki = 77 microM), salsolidine (Ki = 6 microM), and carnegine (Ki = 2 microM). As a class, the 3,4-dihydro-isoquinolines were the most potent inhibitors tested (Ki = 2-130 microM), and the fully aromatic isoquinolines had intermediate activity (Ki = 17-130 microM) against MAO A. In contrast, only a few of these compounds markedly inhibited MAO B. 1,2,3,4-Tetrahydroisoquinoline, its 2-methyl derivative, and o-methylcorypalline gave apparent Ki values of 15, 1, and 29 microM, respectively, and two 3,4-dihydroisoquinolines (compounds 22 and 25) showed substantial inhibition of MAO B (Ki = 76 and 15 microM, respectively). These results support the concept that the topography of the inhibitor binding site differs in MAO A and B.

Alkaloids↗