Biomedical subjects

B E Kaplan

Publications and source records attributed to B E Kaplan.

At least 37 records · Page 2Linked to original sources

PubMed1988

Direct detection of HIV-1 RNA from AIDS and ARC patient samples.

Human immunodeficiency virus (HIV), formerly termed human T-lymphotropic virus (HTLVIII/LAV), is the etiological agent of acquired immune deficiency syndrome (AIDS). Direct detection of HIV-1 nucleic acid sequences in patient tissue or blood samples is possible in only a minor fraction of cases due to the low percentage of infected cells (Shaw et al., 1984). We report a modification of the polymerase chain reaction method (PCR) (Saiki et al., 1985), in which we amplify sequences from HIV-1 RNA templates, for the identification of HIV-1 in peripheral blood and tissue samples obtained from AIDS and AIDS-related complex (ARC) patients. This method of HIV-1 detection is at least six orders of magnitude more sensitive than standard nucleic acid detection methods and has direct clinical applications. In vitro tissue culturing of the virus is not required for HIV-1 detection. Using this technique, the sequence in the orfB region of HIV-1 has been amplified and detected from less than 1 microgram of total RNA prepared from a few milliliters of peripheral blood samples. This technique enables the rapid and unambiguous clinical detection of potential HIV-infected individuals and can be used to assay the efficacy of anti-HIV-1 drugs. To enhance the efficiency of this technique, we have appended the prokaryotic T7 RNA polymerase promoter sequence to one of the priming oligonucleotides. After several cycles of PCR with the promoter-containing oligo, a small aliquot of the reaction can be utilized to direct specific and efficient T7 RNA polymerase-mediated transcription of the amplified sequences, thus enhancing the sensitivity and simplifying the labor of the experiment.

AIDS-Related Complex↗

PubMed1988

Inhibition of human immunodeficiency virus by using an oligonucleoside methylphosphonate targeted to the tat-3 gene.

Antiviral effects were characterized for two oligodeoxyribonucleoside methylphosphonates synthesized in an antisense (3'-TCTTAACC-5') or a sense (5'-AGAATTGG-3') orientation, based on the RNA sequence of the first splice acceptor site of the tat-3 gene of human immunodeficiency virus (HIV) (5'...AGAAUUGG...3'). The development of syncytial cells and supernatant reverse transcriptase was inhibited by a single exposure to the antisense HIV, and HIV RNA synthesis was inhibited by both antisense and sense methylphosphonates but not by a control herpes simplex virus antisense sequence.

Antiviral Agents↗

PubMed1987

Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR.

An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.

Adenine↗

PubMed1987

NMR studies on an oligodeoxynucleotide containing 2-aminopurine opposite adenine.

A heteroduplex containing the mismatch 2-aminopurine (AP)-adenine has been synthesized and studied by proton NMR. The mismatch was incorporated into the sequence d[CGG(AP)GGC].d-(GCCACCG). One-dimensional nuclear Overhauser effect measurements in H2O and two-dimensional nuclear Overhauser effect spectra in D2O show AP.A base pairs in a wobble structure in which both bases are in the anti conformation. The adenine is stacked well in the helix, but the helix twist between the adenine and neighboring cytosine in the 3' direction is unusually small. As a result, the aminopurine on the opposite strand is somewhat pushed out of the helix. From the measurements of the imino proton line widths, the two adjacent G.C base pairs are not found to be significantly destabilized by the presence of the purine-purine wobble pair.

2-Aminopurine↗

PubMed1987

NMR studies of the stable mismatch purine-thymine in the self-complementary d(CGPuAATTTCG) duplex in solution.

One- and two-dimensional nuclear Overhauser effect experiments demonstrate that a single hydrogen bond between a T imino proton and purine N3 is sufficient to hold the base pair dPu.dT in d(CGPuAATTTCG) by a Watson-Crick fashion rather than a Hoogsteen type. In addition, the dPu.dT base pair is well stacked with neighboring base pairs. The spin-lattice relaxation measurements at 30 and 35 degrees C of two decamers, d(CGPuAATTTCG) and d(CGAAATTTCG), reveal that the elimination of two single hydrogen bonds of dA.dT base pairs (due to the substitution of adenine for purine) in the sequence results in an increase in the overall imino proton exchange rate from 7 to 36 s-1 at the site of mismatch.

Base Composition↗

PubMed1987

Nucleotide insertion kinetics opposite abasic lesions in DNA.

A gel assay is introduced to measure DNA polymerase insertion kinetics at single sites along a DNA template strand. The assay is used to analyze the kinetics of inserting deoxynucleotides opposite a synthetic abasic (apurinic/apyrimidinic) lesions using Drosophila DNA polymerase alpha. The location of the abasic lesion next to different nearest-neighbor bases allows the effects of base stacking on the specificity of insertion to be evaluated. The specificity of nucleotide insertion, Vmax/Km, is 6-11 times greater for A over G and about 20-50 times greater for A over C and T. The insertion specificity at the abasic lesion appears to depend more on differences in Vmax than Km. Apparent Michaelis constants for inserting A and G deoxynucleotides are similar to within about a factor of 2. The insertion of A or G occurs most efficiently at the abasic lesion when T is the 5'-nearest neighbor on the primer strand and least efficiently when G is the 5'-nearest neighbor. The presence of different base stacking partners adjacent to the site of insertion has up to a 4-fold effect on specificity.

Animals↗

PubMed1987

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR.

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.

Adenine↗

PubMed1986

Synthesis and properties of oligonucleotides containing 2'-deoxynebularine and 2'-deoxyxanthosine.

The preparation of synthetic oligonucleotides containing 2'-deoxynebularine (dN) and 2'-deoxyxanthosine (dX) is described. The thermal stabilities of duplexes containing dX, dN, and 2'-deoxyinosine (dI) base-paired with the four natural bases have been measured. Xanthine base pairs have stabilities at pH 5.5 that are similar to those of dI-containing duplexes at neutral pH. When xanthine is paired with adenine or cytosine an unusual stabilization of the duplex structure is observed at acid pH. Incorporation of base mispairs opposite template xanthine sites were measured using Drosophila DNA polymerase alpha. The relative nucleoside incorporation rates are in the order: T greater than C much greater than A approximately equal to G. These rates do not correlate with relative thermodynamic stabilities of base mispairs with xanthine obtained from Tm measurements: T greater than G greater than A approximately equal to C. We suggest that DNA polymerase misinsertion rates are greatest when the base mispair can be formed in accordance with Watson-Crick as opposed to other base pairing geometries even though other geometries, e.g. wobble, may result in a more stable final DNA product.

Chemical Phenomena↗

PubMed1986

Synthesis and properties of defined DNA oligomers containing base mispairs involving 2-aminopurine.

DNA heptamers containing the mutagenic base analogue 2-aminopurine (AP) have been chemically synthesized and physically characterized. We report on the relative stabilities of base pairs between AP and each of the common DNA bases, as determined from heptamer duplex melts at 275 and 330 nm. Base pairs are ranked in order of decreasing stability: AP.T greater than AP.A greater than AP.C greater than AP.G. It is of interest that AP.A is more stable than AP.C even though DNA polymerase strongly favors the formation of AP.C over AP.A base pairs. Comparisons of melting profiles at 330 nm and 275 nm indicate that AP.T, AP.A, and AP.C base pairs are annealed in heptamer duplexes and melt 2-3 degrees prior to surrounding base pairs, whereas AP.G appears not to be annealed.

2-Aminopurine↗

PubMed1986

Base pairing and mutagenesis: observation of a protonated base pair between 2-aminopurine and cytosine in an oligonucleotide by proton NMR.

2-Aminopurine (AP), a potent mutagenic base analogue, most frequently pairs with thymine. In the AP X T base pair, both bases adopt normal tautomeric forms. The mechanism for the mutagenic activity arises from its observed pairing with cytosine, which has been ascribed to an enhanced tendency to adopt the rare imino tautomeric form. NMR studies in H2O on all the exchangeable protons in an oligonucleotide duplex containing an AP X T base pair show Watson-Crick hydrogen bonding. When the thymine is replaced by cytosine in the duplex, we observe an AP X C base pair. Both amino protons of AP are seen excluding the rare tautomeric form. Although several alternative structures are possible, it is shown that the second hydrogen bond is formed by protonation of the AP X C base pair and that this is the dominant species under physiological conditions.

2-Aminopurine↗

PubMed1979

Metal-binding and detoxification effect of synthetic oligopeptides containing three cysteinyl residues.

Metallothionein is a naturally occurring metal-binding protein with high cysteine content. Oligopeptides containing three cysteinyl residues and having amino acid sequences analogous to portions of this protein were synthesized by the solid-phase method. Strong affinity of the synthetic peptides to Cd2+ and Zn2+ was observed, and the dissociation constants of the peptide-metal complexes were 2-4 orders of magnitude lower than those of cysteine-metal and dithioerythritol-metal complexes. Effectiveness of detoxification of the peptides against Cd toxicity was demonstrated by the higher survival rates of mice treated with the peptides and by the neutralization of Cd toxicity by the peptides in tissue cultures.

Cations, Divalent↗

PubMed1979

Head and neck cancer: a threat to life and social functioning.

This paper deals with the crisis of cancer and the communication loss faced by the laryngectomized patient. It considers the particular issues for the patient and his family and how the use of crisis intervention can reinforce the coping patterns of the patient and his family.

Adaptation, Psychological↗

PubMed1978

Human blood group glycosyltransferases. I. Purification of n-acetylgalactosaminyltransferase.

An N-acetylgalactosaminyltransferase, which converts blood group O red blood cells to A cells, was purified to homogeneity from plasma of blood group A1 subjects. The enzyme was adsorbed on Sepharose 4B, and after washing out the impurities, the enzyme was eluted with UDP. This procedure resulted in a 70,000- to 100,000-fold increase in specific activity with recovery of about 80%. Further purification of the enzyme was achieved by Bio-Gel P treatment. The final enzyme preparation showed a single protein band, which coincided with enzyme activity, on acrylamide gel electrophoresis, and revealed a single protein band on sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight (90,000 to 100,000), which was estimated by Sephadex gel filtration, and the subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ and had optimum activity at pH 6.5 to 7.0.

ABO Blood-Group System↗

PubMed1973