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Biomedical subjects

K B Jacobson

Publications and source records attributed to K B Jacobson.

At least 19 recordsLinked to original sources
PubMed1998

Product yields from irradiated glycylglycine in oxygen-free solutions: Monte Carlo simulations and comparison with experiments.

The radiation chemistry of photon-irradiated aqueous solutions of biological molecules may be considered under four distinct time regimes: physical transport (< or = 10(-15) s); prechemical conversion of H2O+, H2O*, and subexcitation electrons into free radicals and molecular products (10(-15) s to 10(-12) s); chemical reactions within individual electron tracks (10(-12) s to 10(-6) s); and chemical reactions within overlapping tracks (>10(-6) s). We have previously reported of the use of the Monte Carlo radiation transport/chemistry codes OREC and RADLYS to model the radiolysis of glycylglycine in oxygen-free solution to a time of 1 micros. These simulations successfully predicted the yields of free ammonia, an end product created solely in the reaction of the hydrated electron with the solute within individual tracks. Other measurable products are only partially created during intratrack reactions, and thus one must additionally consider the late, intertrack chemistry of this system. In this paper, we extend our simulations of glycylglycine radiolysis to model for the first time the events which occur during this late chemistry stage. The model considers the product rates of the reactants in bulk solution by using previously available microsecond intratrack yields given by single-track OREC/RADLYS simulations and an x-ray dose rate of 2.80 Gy min(-1) as used in a companion experimental program. These rates are then applied in a series of coupled, differential rate equations that describe the solution chemistry of glycylglycine radiolysis. Product yields are reported as a function of time over a total irradiation period of 10(4) s. Excellent overall agreement is seen between the theoretical predictions and measurements of five radiolysis end products: free ammonia, acetylglycine, diaminosuccinic acid, aspartic acid, and succinic acid. The model also gives the explicit contributions of intratrack and intertrack reactions to the various end products. For example, the model predicts that approximately 56% and 93% of succinic acid and aspartic acid, respectively, are produced during intertrack reactions at a solute concentration of 0.05 M; these contributions drop to 0.07% and 11%, respectively, at 1.2 M.

Computer Simulation↗
PubMed1997

Analysis of biosensor chips for identification of nucleic acids.

Two novel DNA-sequencing methods are described that use DNA hybridization biosensor chips. These two techniques involve either labeling the free nucleic acid with enriched stable isotopes or hybridizing DNA without labels to immobilized peptide nucleic acid (PNA) and detecting the phosphorus present in the DNA but not in the PNA. Sputter-initiated resonance ionization microprobe analysis was used to detect the presence of enriched tin isotope-labeled DNA and of phosphorus in natural DNA as a means to identify the presence of DNA after hybridization to oligodeoxynucleotides (ODNs) or PNAs, respectively, immobilized on a biosensor chip. The data clearly demonstrate that excellent discrimination between complementary and noncomplementary sequences can be obtained during hybridization of DNA to either ODNs or PNAs. The capability to detect different enriched stable isotope-labeled DNAs simultaneously allows high degrees of multiplexing which may be very advantageous for hybridization kinetics studies in complex systems, as well as significantly increasing the speed of analysis. Alternatively, by using natural DNA with PNA biosensor chips, discrimination for single-point mutation could be increased because of improved hybridization kinetics and direct analysis of genomic DNA may become possible without amplification. Both methods have the potential to provide a rapid method for DNA/RNA sequencing, diagnostics, and mapping.

Biosensing Techniques↗
PubMed1997

Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels.

A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched in isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method.

DNA↗
PubMed1995

Analysis of polymerase chain reaction-amplified DNA products by mass spectrometry using matrix-assisted laser desorption and electrospray: current status.

Recent advances in molecular biology are making it possible to diagnose genetic diseases and identify pathogens through the analysis of DNA. As clinical applications for molecular diagnosis increase, rapid, reliable methods for determination of DNA size will be needed. Mass spectrometry offers the potential of analyzing amplified DNA quickly and reliably, without the need for gel-based separation and sample labeling steps that are conventionally employed. Both electrospray ionization and matrix-assisted laser desorption/ionization have been evaluated for the size analysis of DNA using both synthetic oligonucleotides and PCR-amplified samples corresponding to bases 1626 to 1701 of the cystic fibrosis transmembrane conductance regulator gene. Both technologies have been demonstrated to have mass range and sensitivity required for the analysis of PCR-amplified DNA in this size range using minimal sample preparation. Steps required to incorporate either ionization technique into a reliable analytical scheme for the rapid, routine analysis of DNA are outlined.

Base Sequence↗
PubMed1995

Optical melting of 128 octamer DNA duplexes. Effects of base pair location and nearest neighbors on thermal stability.

The use of short oligonucleotide probes is finding increased application in DNA sequencing and genome characterization techniques, but a lack of knowledge of the hybridization properties of short duplexes hinders their use. Melting data were acquired on 128 DNA duplexes based on the length proposed in sequencing by hybridization procedures and formed from the general sequences 5'-XYZTGGAC-3',5'-GTCCAXYZ-3',5'-GCXYZGAC-3', and 5'-GTCXYZGC-3' where X, Y, and Z are either A, T, G, or C. These molecules were designed to elucidate the effects of location and nearest-neighbor stacking on the stability of base pairing in short DNA duplexes. The type of base pairs present had a major effect on stability, but was insufficient to predict stability without the inclusion of nearest-neighbor terms. Furthermore, the addition of information on position, or distance from the end, of the nearest-neighbor doublets led to statistically better fitting of the melting data. However, the positionally dependent stabilization differences are small compared with the contributions of base pairing and stacking.

Base Composition↗
PubMed1994

Picolinic acid as a matrix for laser mass spectrometry of nucleic acids and proteins.

We found that picolinic acid is a very good matrix for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry of oligonucleotides, proteins and tRNA. Among the oligonucleotides, picolinic acid was shown to be effective for homo-oligonucleotides, d(G)40 and d(C)60, and for mixed-base oligonucleotides up to 190 bases. In the case of the single-stranded oligonucleotides, and of double-stranded ones as well, only parent ions corresponding to single-stranded DNA were observed. The efficiency for MALDI of oligonucleotides using the picolinic acid matrix was superior to that using 3-hydroxypicolinic acid. MALDI of transfer RNA phenylalanine-specific (tRNA(Phe)), a 76-base ribonucleic acid, was detected with a signal-to-noise ratio of > 10. By comparison with 3-hydroxypicolinic acid the results with picolinic acid are notably superior for all oligonucleotides.

Base Sequence↗
PubMed1993

Use of resonance ionization spectroscopy to detect DNA bands on ultrathin spin-coated gels.

Development of alternative electrophoresis procedures are necessary for large volume sequencing and mapping studies. The use of stable isotopes as DNA labels and ultrathin gels promises to greatly increase the rate of sequencing. Spin coating is presented as an alternative method for producing ultrathin polyacrylamide gels. The technique has the potential of producing gels of micron to submicron thicknesses by varying the viscosity of the acrylamide solution and the spinning speed. Thirty micron thick 6% (weight %) gels were produced in this manner. Tin-labeled DNA oligomers were electrophoresed and detected using sputter-initiated resonance ionization spectroscopy (SIRIS). The usefulness of SIRIS and laser atomization RIS (LARIS) to sample the surface and deeper layers of 240 microns thick gels was investigated. With LARIS, whole cross-sections of the gel can be atomized, possibly allowing complete sampling of labels.

DNA↗
PubMed1992

Immobilization of DNA for scanning probe microscopy.

Reproducible scanning tunneling microscope and atomic force microscope images of entire molecules of uncoated plasmid DNA chemically bound to surfaces are presented. The chemically mediated immobilization of DNA to surfaces and subsequent scanning tunneling microscope imaging of DNA molecules demonstrate that the problem of molecular instability to forces exerted by the probe tip, inherent with scanning probe microscopes, can be prevented.

DNA, Bacterial↗
PubMed1992

Electrophoresis and detection of tin-labeled DNAs on open-faced gels.

Alternative protocols are necessary for the use of polyacrylamide gel electrophoresis in genome scale sequencing and mapping studies. The use of radioisotopes and manual gel reading will have to be replaced with a flexible labeling system that can be detected at levels similar or to better than radioisotopes but allows automated, high-speed detection. Labeling with stable isotopes is such an alternative. These nondecaying isotopes have the potential to be detected in sub-attomole quantities, despite being surrounded by the gel matrix, due to the high selectivity and sensitivity of resonance-ionization spectroscopy coupled with a mass spectrometer. In this study the detection limits of sputter-initiated resonance ionization spectroscopy (SIRIS) are investigated using thin, open-faced polyacrylamide gels supported by plastic. This system allows reproducibility and flexibility in the choice of gel size and buffer system since the gel can be cast, washed free of polymerization by-products, dried, and stored until use. Various concentrations of an Sn-labeled oligomer were run on these gels and loads of 5 femtomoles/mm could be detected on a 240 microns thick gel. Gels as thin as 60 microns lower the detectable concentration loads to 1 femtomole/mm. The limiting factor is tin contamination in the gel which, if reduced, will further increase detection. Polymerase chain reaction (PCR) products can also be labeled and detected using Sn isotopes, which could prove useful in mapping studies. Also presented are techniques which will facilitate resolution of these PCR products on open-faced gels by employing discontinuous buffers systems and DNA mobility modifiers.

Chromosome Mapping↗
PubMed1992

Scanning tunneling microscopy of DNA: a novel technique using radiolabeled DNA to evaluate chemically mediated attachment of DNA to surfaces.

pBS+ plasmid deoxyribonucleic acid (DNA) was imaged by scanning tunneling microscopy (STM) after mounting microdroplets by aerosol deposition onto heated epitaxial gold surfaces. However, the instability of the adsorbate to forces exerted by the tunneling tip points out the need for more aggressive bonding of molecules to surfaces. We describe a sensitive assay for the qualitative and quantitative evaluation of chemical agents to influence binding of DNA to surfaces using 32P-labeled pBS+ plasmid DNA. We propose that such an assay can make an important contribution to immobilization techniques prior to STM imaging.

DNA↗
PubMed1992

A comparative study of Drosophila phenylalanine hydroxylase with a natural and a synthetic tetrahydropterin as cofactor.

1. Phenylalanine hydroxylase activity has been analyzed in Drosophila melanogaster using as cofactors the natural tetrahydropteridine 5,6,7,8-tetrahydrobiopterin (H4Bip) and the synthetic one 5,6-dimethyl-5,6,7,8-tetrahydropterin (H4Dmp). 2. The apparent Vmax and KM for substrate and cofactor showed that the enzyme has two times more affinity for the substrate when H4Bip is the cofactor in the reaction. Similarly to what was found with purified rat liver phenylalanine hydroxylase, H4Bip was the most effective cofactor, leading to 4-5 times more activity than that obtained with H4Dmp. 3. With the natural cofactor H4Bip, no activation of the enzyme with Phe was necessary (in contrast to mammalian phenylalanine hydroxylase), and this tetrahydropteridine inhibits phenylalanine hydroxylase activity when the enzyme is exposed to it before phenylalanine addition. With the synthetic H4Dmp, both types of preincubations led to an increase of phenylalanine hydroxylase activity. 4. The enzyme is highly unstable compared to mammalian phenylalanine hydroxylase, even at -20 degrees C. 5. Thorax and abdomen extracts caused significant inhibition of phenylalanine hydroxylase activity from third instar larvae or newborn adult head extracts, when assayed with the synthetic cofactor H4Dmp. This inhibition did not happen with H4Bip. The presence of the pteridine 7-xanthopterin in adult bodies was not the cause of this inhibition.

Animals↗
PubMed1992

Atomic force microscopy of DNA on mica and chemically modified mica.

Atomic force microscopy (AFM) was used to image circular DNA adsorbed on freshly cleaved mica and mica chemically modified with Mg(II), Co(II), La(III), and Zr(IV). Images obtained on unmodified mica show coiling of DNA due to forces involved during the drying process. The coiling or super twisting appeared to be right handed and the extent of super twisting could be controlled by the drying conditions. Images of DNA observed on chemically modified surfaces show isolated open circular DNA that is free from super twisting, presumably due to strong binding of DNA on chemically modified surfaces.

Adsorption↗
PubMed1992

Measurement of products from X-irradiated glycylglycine in oxygen-free aqueous solutions.

The product yields in X-irradiated aqueous solutions of glycylglycine (0.05 M and 1.0 M) were measured under deoxygenated conditions. Comparison was made between the results obtained from X- and 60Co gamma-irradiated glycylglycine solutions reported by Garrison, Sokol, and Bennett-Corniea (Radiat. Res. 53, 376-384, 1973). The mechanisms proposed by Garrison et al. were tested by evaluating the stoichiometric relationships. The two intermediate radicals, deamination and H-abstraction radicals, were produced in the initial interactions of glycylglycine with reactive species (e-aq, OH, H) formed in H2O. Although the difference was fairly large at 0.05 M, the production of deamination radicals agreed well with the consumption of the radicals at 1.0 M. The production and the consumption of H-abstraction radicals were within the estimated experimental error in dilute solutions. Among all the products only the G value of aspartic acid decreased with increasing concentration of glycylglycine. This could be attributed to the fact that more acetylglycine is formed at the expense of aspartic acid at 1.0 M than at 0.05 M glycylglycine solutions. Competitive reactions involved with deamination radicals under conditions of homogeneously distributed reactants are discussed to elucidate the radiation chemistry of glycylglycine.

Cobalt Radioisotopes↗
PubMed1991

Isolation and characterization of GTP cyclohydrolase I from mouse liver. Comparison of normal and the hph-1 mutant.

GTP cyclohydrolase I, an enzyme that catalyzes the first reaction in the pathway for the biosynthesis of pterin compounds, was purified from of C3H mouse liver by 192-fold to apparent homogeneity, using Ultrogel AcA34, DEAE-Trisacryl, and GTP-agarose gels. Its native molecular weight was estimated at 362,000. When the enzyme was subjected to electrophoresis on a denaturing polyacrylamide gel, only one protein band was evident, and its molecular weight was estimated at 55,700. The NH2-terminal amino acid of this enzyme was serine. These results indicate the enzyme consists of six to eight subunits. No coenzyme or metal ion was required for activity. This enzyme activity was inhibited by most of divalent cations and was slightly activated by potassium ion. The Km value for GTP was determined to be 17.3 microM. The temperature and pH optima for the activity were 60 degrees C and pH 8.0-8.5, respectively. The expected products, a dihydroneopterin compound and formic acid, were found in a molar ratio of 1.01. A polyclonal antiserum generated against the purified enzyme was used to compare GTP cyclohydrolase I from the hph-1 mutant and normal mouse. The hph-1 mutant liver contained only 8% of normal specific activity, but a normal amount of GTP cyclohydrolase I antigen as compared with the C3H mouse. Subunit molecular weight and electrophoretic behavior of GTP cyclohydrolase I from hph-1 mutant were not different from those of the enzyme from C3H mouse. These results suggest that the hph-1 mutation may involve alteration of the catalytic site but does not detectably alter the whole enzyme structure.

Animals↗
PubMed1991

Strontium toxicity in Drosophila melanogaster.

The Drosophila strains v;bw and Austin represent the extremes in resistance and sensitivity, respectively, to the toxic effects of CdCl2 in the diet. Using the six genotypes containing the possible combinations of the three main chromosomes for v;bw and Austin, we had shown previously that the gene(s) for cadmium resistance is on the X-chromosome. In this paper it is shown, using the same six genotypes, that the gene(s) for strontium resistance is on the second chromosome, as measured by LC50 values.

Animals↗
PubMed1991

Electrochemically induced adsorption of radio-labeled DNA on gold and HOPG substrates for STM investigations.

In a scanning tunneling microscope (STM) electrochemical cell we have studied the effects of electrode potential on both the surface topography and the adsorption of deoxyribonucleic acid (DNA) to graphite and gold surfaces. Images of the surface of highly oriented pyrolytic graphite (HOPG), of the same area, in response to a positive increase in surface potential show degradation of the step edges with little change in the crystal plane. Images of the same area of a gold surface demonstrate the formation of and the progressive increase in nodular structures on the crystal planes, in response to increased potential, with little effect on the step edges. Using radio-labeled DNA we monitored electrochemical absorption onto HOPG and gold surfaces. Although at no applied potential and at negative surface potentials some DNA was bound, at positive potentials 3 to 5 times more DNA was incorporated onto both surfaces. DNA adsorbed to a surface at a positive potential was not removed by reversing the potential.

Adsorption↗
PubMed1991

Radiation damage to a biomolecule: new physical model successfully traces molecular events.

For the first time, a complete computer simulation of physical and chemical reactions at the molecular level has been used to calculate the yield of a chemical species resulting from irradiation of a biological molecule in aqueous solution. Specifically, when a solution of glycylglycine is irradiated anaerobically, an ammonia molecule is released by the action of a hydrated electron, which is produced by irradiation of water. In the computations, Monte Carlo techniques are used to simulate the statistical progression of molecular events as they are assumed to occur. These include the initial physical ionization and excitation of water molecules along a particle track in the liquid; the subsequent formation of free radicals and other species: and the random diffusion and chemical reactions of the species with each other, the solvent, and solute molecules. We have calculated and measured the yield of ammonia from irradiation of glycylglycine with 250 kVp x-rays as a function of glycylglycine concentration between 0.01 and 1.2 M. Excellent agreement is obtained between predicted and measured results. The literal simulation of events, combined with specific experimental measurements, offers a powerful new tool for studying mechanisms of radiation action and damage at the molecular level.

Ammonia↗