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Biomedical subjects

L J Arnold

Publications and source records attributed to L J Arnold.

At least 19 recordsLinked to original sources
PubMed2001

DNA exhibits multi-stranded binding recognition on glass microarrays.

In the course of exploring the hybridization properties of glass DNA microarrays, multi-stranded DNA structures were observed that could not be accounted for by classical Watson-Crick base pairing. Non-denatured double-stranded DNA array elements were shown to hybridize to single-stranded (ss)DNA probes. Similarly, ssDNA array elements were shown to bind duplex DNA probes. This led to a series of experiments demonstrating the formation of multi-stranded DNA structures on the surface of microarrays. These structures were observed with a number of heterogeneous sequences, including both purine and pyrimidine bases, with shared sequence identity between the ssDNA and one of the duplex strands. Furthermore, we observed a strong binding preference near the ends of duplexes containing a 3'-homologous strand. We suggest that such binding interactions on cationic solid surfaces could serve as a model for a number of biological processes mediated through multi-stranded DNA.

DNA↗
PubMed2001

Optimized T7 amplification system for microarray analysis.

Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).

Biotechnology↗
PubMed1996

Synthesis and thermodynamics of oligonucleotides containing chirally pure R(P) methylphosphonate linkages.

Methylphosphonate (MP) oligodeoxynucleotides (MPOs) are metabolically stable analogs of conventional DNA containing a methyl group in place of one of the non-bonding phosphoryl oxygens. All 16 possible chiral R(P) MP dinucleotides were synthesized and derivatized for automated oligonucleotide synthesis. These dimer synthons can be used to prepare (i) all-MP linked oligonucleotides having defined R(P) chirality at every other position (R(P) chirally enriched MPOs) or (ii) alternating R(P) MP/phosphodiester backbone oligonucleotides, depending on the composition of the 3'-coupling group. Chirally pure dimer synthons were also prepared with 2'-O-methyl sugar modifications. Oligonucleotides prepared with these R(P) chiral methylphosphonate linkage synthons bind RNA with significantly higher affinity than racemic MPOs.

Chromatography, High Pressure Liquid↗
PubMed1996

Antisense oligonucleotide containing an internal, non-nucleotide-based linker promote site-specific cleavage of RNA.

We have designed and synthesized a series of novel antisense methylphosphonate oligonucleotide (MPO) cleaving agents that promote site-specific cleavage on a complementary RNA target. These MPOs contain a non- nucleotide-based linking moiety near the middle of the sequence in place of one of the nucleotide bases. The region surrounding the unpaired base on the RNA strand (i.e. the one directly opposite the non-nucleotide-linker) is sensitive to hydrolytic cleavage catalyzed by ethylenediamine hydrochloride. Furthermore, the regions of the RNA comprising hydrogen bonded domains are resistant to cleavage compared with single-stranded RNA alone. Several catalytic moieties capable of supporting acid/base hydrolysis were coupled to the non-nucleotide-based linker via simple aqueous coupling chemistries. When tethered to the MPO in this manner these moieties are shown to catalyze site-specific cleavage on the RNA target without any additional catalyst.

Base Sequence↗
PubMed1994

Triple-strand-forming methylphosphonate oligodeoxynucleotides targeted to mRNA efficiently block protein synthesis.

Antisense oligonucleotides are ordinarily targeted to mRNA by double-stranded (Watson-Crick) base recognition but are seldom targeted by triple-stranded recognition. We report that certain all-purine methylphosphonate oligodeoxyribonucleotides (MPOs) form stable triple-stranded complexes with complementary (all-pyrimidine) RNA targets. Modified chloramphenicol acetyltransferase mRNA targets were prepared with complementary all-pyrimidine inserts (18-20 bp) located immediately 3' of the initiation codon. These modified chloramphenicol acetyltransferase mRNAs were used together with internal control (nontarget) mRNAs in a cell-free translation-arrest assay. Our data show that triple-strand-forming MPOs specifically inhibit protein synthesis in a concentration-dependent manner (> 90% at 1 microM). In addition, these MPOs specifically block reverse transcription in the region of their complementary polypyrimidine target sites.

Base Sequence↗
PubMed1993

Deprotection of methylphosphonate oligonucleotides using a novel one-pot procedure.

Deprotection of methylphosphonate oligonucleotides with ethylenediamine was evaluated in a model system. Methylphosphonate sequences of the form 5'-TTTNNTTT, where N was either N4-bz-dC, N4-ibu-dC, N2-ibu-O6-DPC-dG, N2-ibu-dG, N6-bz-dA, or T, were used to determine the extent of modifications that occur during deprotection. Up to 15% of N4-bz-dC was found to transaminate at the C4 position when treated with ethylenediamine. A similar displacement reaction with ethylenediamine was observed at the O6 position of N2-ibu-O6-DPC-dG, and to a much lesser extent of N2-ibu-dG. Side reactions were not observed when oligonucleotides containing N4-ibu-dC, N6-bz-dA, or T were treated with ethylenediamine. A novel method of deprotecting methylphosphonate oligonucleotides was developed from these studies. The method incorporates a brief treatment with dilute ammonia for 30 minutes followed by addition of ethylenediamine for 6 hours at room temperature to complete deprotection in a one-pot format. The solution is then diluted and neutralized to stop the reaction and prepare the crude product for chromatographic purification. This method was used to successfully deprotect a series of oligonucleotides at the 1, 100, and 150 mumole scales. These deprotection results were compared to a commonly used two-step method and found to be superior in yield of product by as much as 250%.

Ammonia↗
PubMed1993

An improved method for the synthesis and deprotection of methylphosphonate oligonucleotides.

The methylphosphonate oligonucleotide synthesis methods described here give the desired products in good yield. Superior amounts of product are achieved by modifying both the DNA synthesis program and the reagent to compensate for the unstable methylphosphonite intermediate. Deprotection conditions have also been altered to maximize the recovery of oligonucleotide from DNA synthesis supports and to minimize the amount of base modification. Mass-spectrometry analysis of our oligonucleotides has verified their purity and confirmed the absence of modified bases. When compared to standard DNA synthesis methods, this procedure uses only about one-third the usual amount of monomer. Using these procedures, it should be possible to synthesize reliably methylphosphonate oligonucleotides at 1- and 15-mumol scales.

Base Sequence↗
PubMed1990

A scale model study of the effects of meteorological, soil, and house parameters on soil gas pressures.

To assess the contribution of various environmental parameters to the entry of Rn into basements, it is advantageous to simplify and control the important variables present in the field situation. A scale model system, simulating a house in soil, has been constructed to investigate the effect of meteorological parameters and house and soil characteristics on soil gas flow around houses. The house walls and soil are of variable permeability. Wind is simulated by applying a static pressure distribution to the soil surface. The effect of temperature differences and appliances is simulated by depressurizing the model house. Soil gas pressures at various locations around the house are measured under different conditions. The results show that the ratio of wall to soil permeability is the determining factor in soil gas flow patterns. For a wind of 8.94 m s-1 (20 mph), the horizontal pressure gradients are about 99 Pa m-1 in the model when the wall is at least as permeable as the soil. This corresponds to 3.3 Pa m-1 in the field. When the soil is two or more orders of magnitude more permeable than the wall, the gradient is about 19.8 Pa m-1 in the model, or 0.66 Pa m-1 in the field. There is a logarithmic dependence of pressure gradient on the ratio of wall to soil permeability in the range -2 less than log (kw/ks) less than 0. Conversely, it takes a large temperature difference of 27 degrees C to cause a 99 Pa m-1 horizontal pressure gradient in model systems with wall permeability greater than soil permeability. The effects of changes in the model system on soil gas flow patterns are investigated for the cases of lowered soil surface permeability, partial surface capping, and presence of a subfloor gravel bed. Partial surface capping, as would occur with driveways and patios, was found to have a minor effect on soil gas pressures. However, lowered surface permeability, caused by precipitation, can significantly change soil gas flow patterns. The only change in soil pressure gradients or pressure differences in the presence of a gravel bed is in the system with the highest wall-to-soil permeability ratio. In this system, under all conditions (house depressurization, wind, and wind with house depressurization), there is an increase in the absolute value of both upwind and downwind pressure differences and pressure gradients with the addition of a gravel bed.

Air Pollutants↗
PubMed1989

Assay formats involving acridinium-ester-labeled DNA probes.

We describe the development of several hybridization assay formats involving acridinium-ester-labeled DNA probes. The simplest of these is a homogeneous assay procedure that requires only three steps to complete, including a 5-s detection step. Using this format, we have detected target sequences in the 10(-16) to 10(-17) mol range; when rRNA is the target, this translates to 3000 to 300 bacterial organisms. The entire assay can be carried out in less than 30 min. This is the first homogeneous DNA probe assay to be of practical use in the clinical laboratory, and it represents a major simplification of hybridization formats. We also demonstrate the use of this homogeneous assay format to discriminate single-base differences between two closely related target sequences and to detect DNA as well as RNA target molecules. By combining homogeneous hybrid discrimination with solid-phase separation, we have been able to decrease background readings from unhybridized probe to only a few parts per million. This enhances assay sensitivity about 10-fold, to a range of 10(-17) to 10(-18) mol of target. We are in the process of further improving the performance of these assays.

Acridines↗
PubMed1984

Inhibition by magnesium and calcium acetates of lead subacetate- and nickel acetate-induced lung tumors in strain A mice.

The ability of the physiologically essential divalent metals calcium and magnesium to inhibit the tumorigenic activities of lead and nickel towards the lungs of strain A mice was investigated. The tumorigenic salts lead(II) subacetate and nickel(II) acetate were injected i.p. at their maximal tolerated doses (0.04 mmol/kg/injection of each metal) for a total of 24 injections, whenever possible. Calcium(II) acetate and magnesium(II) acetate were administered in the same preparation along with the lead and nickel salts at molar doses of approximately 1, 3, 10, and 30 times the maximal tolerated dose of the tumorigen. The animals were sacrificed 30 weeks after the first injection, and the lung tumors were counted. The lead and nickel salts, administered alone, each produced a significant increase in the observed number of lung adenomas per mouse. When administered with any of the doses of calcium acetate or magnesium acetate tested, neither lead subacetate nor nickel acetate showed any significant tumorigenic activity. Calcium acetate alone (total dose, 11 mmol/kg of body weight) appeared to yield a significant rise in lung adenomas observed. The results indicate an antagonism between magnesium and calcium and the tumorigenic metals nickel and lead.

Acetates↗
PubMed1980

Effect of commercial saccharin preparations on urethan-induced lung tumorigenesis in strain A mice.

The effect of commercial saccharin preparations on urethan-induced mouse lung tumorigenesis was assessed by gavaging groups of male strain A mice with 1-g/kg doses of each saccharin preparation on a daily basis 5 days/week. Gavage was initiated 1 week before i.p. injection of either a low (0.1 mg/g) or a high (1 mg/g) dose of urethan and continued until the mice were sacrificed 16 weeks after urethan administration. The average number of surface lung tumors per mouse for each group of mice was determined and was compared statistically with the appropriate control group. The commercial saccharin preparations did not produce an elevated lung tumor response when administered alone. One of the four saccharin preparations enhanced the lung tumor response to urethan when given in conjunction with the low dose of urethan, but this enhancement was not statistically significant. At the high urethan dose, all saccharin preparations produced a statistically significant enhancement of the lung tumor response to urethan.

Adenoma↗
PubMed1979

Proton nuclear magnetic resonance study of the conformation and configuration of the cyclized pyridine nucleotide adducts.

We have closely examined by high-frequenty 1H nuclear magnetic resonance spectroscopy the structure of the adducts which form when various carbonyl compounds react with pyridine nucleotides at elevated pH. These studies show that the adducts of N-(2,6-dichlorobenzyl)nicotinamide-acetone, n-(2,6-dichlorobenzyl)nicotinamide-pyruvate, NMN-pyruvate, NAD-pyruvate, NAD-acetaldehyde, and NAD-oxaloacetate form with identical structural features as well as configuration. The following structural features are observed: (1) the adducts are pyridine N-4-substituted compounds; (2) a second six-membered ring forms by addition of the nicotinamide amido to the carbonyl group of the compound forming the addition complex; (3) cyclization occurs stereospecifically, indicating that the stereochemistry is predetermined by the initial attack at the N-4 position; (4) two diastereomeric forms are observed for each nucleotide adduct. Finally, the determination of configuration at all symmetric carbon atoms in these adducts will be discussed.

Acetaldehyde↗
PubMed1979

Antineoplastic activity of poly(L-lysine) with some ascites tumor cells.

We have found that poly(L-lysine) can be a very effective agent in preventing the growth of Ehrlich ascites tumors in mice. When given optimal doses of poly(L-lysine) (Mr 60 x 10(3)) intraperitoneally for 5 consecutive days, beginning on day 1 after inoculation with Ehrlich ascites cells. White Swiss mice show nearly a 100% remission from subsequent tumor growth. Rechallenge of "cured" animals with tumor cells, however shows no long-term immunological protection. In tissue culture, poly(L-lysine) shows a related potent cytotoxicity with HeLa cells; interestingly, the D isomer. In addition, there is a strong molecular weight dependence in that the small polylysine (Mr 3 x 10(3)) possesses less than 1/20th the cytotoxicity of large polymers (Mr 70 x 10(3)) on a weight basis in both cell culture and animal studies. At the same time, none of these lysine polymers gives any significant increase in life span to BDF1 mice infected with L1210 murine leukemia cells. We have also further explored the mechanism by which the polylysines express their cytotoxicity. These data indicate that lysine polymers show cell specificity in their action and in some cases they may be beneficial as potent antineoplastic agents, particularly when molecular weight is taken into consideration.

Animals↗