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Biomedical subjects

D Gillespie

Publications and source records attributed to D Gillespie.

At least 19 recordsLinked to original sources
PubMed1992

An hypothesis concerning optimal therapy in HIV disease.

Ampligen, a mismatched double stranded RNA, is hypothesized to be an ideal base therapy for HIV disease to which other agents, such as the nucleoside analogue, AZT, can be advantageously added. The unique properties of Ampligen which support this hypothesis include activation of immune cells, inhibition of virus replication by inducing an antiviral cellular state and inhibition of growth of neoplastic cells. Ampligen is synergistic with other agents being used or being tested for use in HIV disease and is without toxicity.

Adjuvants, Immunologic↗
PubMed1992

Recovering relationships: a feminist analysis of recovery models.

Two models of recovery based on the concepts of independence and interdependence are contrasted from a feminist perspective. Drawing on social and psychological analyses, the authors critique the overemphasis of independence as the goal of health care and instead advocate a more relational model of therapy that reinforces social and emotional connections between people. Two narratives from occupational therapy are used to illustrate the differing assumptions underlying these models. The authors discuss some of the structural and interactional barriers to the expression of interdependence in health care institutions. They suggest that through purposefully sharing practice experiences and instating collaborative, nonhierarchical models of organization, practitioners can begin to overcome these barriers.

Aged↗
PubMed1991

High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.

Animals↗
PubMed1991

Characterization of polyamino acids by use of GPC-viscometry technology.

The properties of polybenzyl glutamate, a well known polyamino acid vary considerably with molecular weight in both solid state and in solution. Therefore, accurate determinations of the molecular weights of these polyamino acids is essential. The dual viscometer/refractometer when used as detector system for size exclusion chromatography provides a way of determining accurate molecular weights. An indirect method of determining the molecular weight distribution (MWD) and the radius of gyration distribution (RgD) of polybenzyl glutamate is described. The MWD is calculated from the measured value of intrinsic viscosity (IV) and the known IV-to-MW relationship, at every SEC retention volume slice. Such a technique of determining MWD requires no calibration and is more precisely measurable than conventional SEC methods.

Amino Acids↗
PubMed1991

High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides.

Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. "Iodopride" (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with [125I]iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, "iclopride" (KD 0.23 nM) and "itopride" (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, "epidepride" (KD 0.057 nM) and "ioxipride" (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8.

Animals↗
PubMed1990

Current concepts in quantitative molecular hybridization.

Knowledge of the molecular biology of cancer and other diseases has grown enormously in the past decade. The role certain genes play in disease states is clearer, creating new opportunities for diagnostic applications of molecular biological techniques in clinical medicine. The measurement of an abundance of certain nucleic acids could be valuable as a diagnostic or prognostic test. For example, aberrant expression of growth-promoting genes has correlated with the type and severity of certain neoplasias. Also, the determination of an abundance of exogenous sequences, arising from viruses or other pathogens, can be useful for evaluating virus load and activity during infection. Changes in the levels of these sequences could provide a means to determine the efficacy of therapy. Nucleic acid sequences can be measured by molecular hybridization using complementary nucleic acids as probes. Strategies for performing quantitative molecular hybridization, and their application in the clinic, are discussed.

DNA↗
PubMed1990

The magic and challenge of DNA probes as diagnostic reagents.

Molecular basis of hybridization and effect of different conditions, such as probe size, salt concentration, temperature, detergent concentration, time of washing, sample preparation and sample purity, on hybridization is discussed. Different formats of hybridization such as in solution hybridization, solid phase hybridization, and methods for probe preparation are presented. Polymerase chain reaction for the amplification of target and alternative methods for signal amplification are introduced in this review.

Blotting, Southern↗
PubMed1989

Definition of a second dimeric subfamily of human alpha satellite DNA.

We describe a new human subfamily of alpha satellite DNA. The restriction endonuclease XbaI cleaves this subfamily into a collection of fragments which are heterogeneous with respect to size. We compared the sequences of 6 clones from four different XbaI size classes. Clones from a single size class were not necessarily more related than clones from different classes. Clones from different size classes were found to produce almost identical hybridization patterns with XbaI-digested human genomic DNA. All clones were found to share a common dimeric repeat organization, with dimers exhibiting about 84% sequence identities, indicating that the clones evolved from a common progenitor alphoid dimer. We show that this subfamily, and the EcoRI dimer subfamily originally described by Wu and Manuelidis, evolved from different progenitor alphoid dimers, and therefore represent distinct human alphoid subfamilies.

Base Sequence↗
PubMed1989

Quantitation of HIV-1 RNA in blood cells of ARC and AIDS patients.

Peripheral blood mononuclear cells from HIV-1-infected persons were mixed with 5 M guanidine thiocyanate, and HIV-1-specific probes were hybridized with target RNA directly in the lysate. No RNA purification was needed. Hybrids were purified by repeated capture on superparamagnetic beads coated with oligo(dT) in a device-assisted format, a procedure termed "reversible target capture." Blood mononuclear cells from 70 ARC and AIDS patients were examined and found to have an average of 1.8 x 10(5) molecules of HIV-1 RNA per 2 x 10(6) cells.

AIDS-Related Complex↗
PubMed1989

A noise-free molecular hybridization procedure for measuring RNA in cell lysates.

A solution hybridization technique was designed to measure RNA abundance in crude cell lysates and at the same time to maximize confidence that signals resulted from true molecular hybridization. Cell lysates were prepared in 5 M guanidine thiocyanate, then RNA molecules in the lysates were hybridized with two probes, a 32P-labeled RNA "label probe" which provided signal and an oligodeoxyribonucleotide "capture probe" containing a poly(dA) tail which provided a mechanism for selective purification. Ternary hybrids were "captured" on oligo(dT)-coated superparamagnetic beads through a readily reversible interaction with the poly(dA) of the capture probe. RNA did not bind to dT beads through poly(A) under the capture conditions used. Hybrids were purified through cycles of capture on and release from dT beads, with each cycle yielding a 100- to 1000-fold reduction in noise (unhybridized label probe) and a 50-90% recovery of signal (hybridized label probe). Noise was driven below detectable limits after three cycles of capture, thereby improving the sensitivity of measuring target RNA. As few as 15,000 target molecules, 15 fg of a 3-kb RNA, was detectable in the equivalent of 2 x 10(6) cells in concentrated cell lysates (10(8) cells/ml). Since hybridization with both probes was required in order to yield a signal, hybridization specificity could be adjusted with either or both probes. The greater specificity and lack of noise increased confidence that the signal was proportional to the amount of RNA of interest.

Guanidines↗
PubMed1989

Probes for quantitating subpicogram amounts of HIV-1 RNA by molecular hybridization.

A set of probes was designed for the quantitation of HIV-1 RNA in infected cells by a molecular hybridization procedure called reversible target capture. Reversible target capture is analogous to sandwich hybridization, except that the link between hybrid complexes and the affinity support was reversible, allowing for repeated capture of hybrids on, and release from, fresh affinity support. Repeated cycles of capture resulted in a high degree of purification of hybrids from unreacted probe, thereby greatly reducing assay noise and increasing assay sensitivity. Probes against the HIV-1 pol gene were chosen because their target sequences were highly conserved among HIV-1 isolates, while being divergent enough to provide discrimination from other human T cell tropic viruses. Subpicogram quantities of HIV-1 pol gene RNA were measured with signal:noise ratios of over 10. Hybridization signal increased with increasing target RNA with a proportionality constant of 1.

Cells, Cultured↗
PubMed1989

Low background scintillation counting.

Counting radioactive samples with Beckman Instrument's Ready Caps, using a restricted energy window, LL-UL = 400-1000, resulted in machine backgrounds of under 2 cpm and efficiencies of counting relative to liquid scintillation cocktails (LSC) of 51%, 65%, 57%, 62%, and 1% for 32P, 125I, 14C, 35S and 3H, respectively. Signal-to-noise ratios from a quantitative molecular hybridization technique were increased 8-10 fold. There may be a general application for this product in experiments yielding low amounts of radioactivity in liquid samples.

Animals↗
PubMed1989

Enzymatic amplification of RNA purified from crude cell lysate by reversible target capture.

For increased clinical applications of nucleic acid probes to gene diagnosis, current procedures must be modified to become more amenable to the rapid processing of many samples with as few manipulations as possible. Here we summarize progress in the development of a strategy for performing molecular hybridization directly in lysate of biological samples dissolved in solutions containing the chaotropic agent, guanidine thiocyanate. Hybrids are purified by a process referred to as "reversible target capture," in which specific nucleic acid sequences are rapidly purified from crude lysate. We illustrate the use of this strategy to assay HIV-1 RNA and to rapidly purify HIV-1 RNA before enzymatic amplification by the polymerase chain reaction method.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Molecular hybridization with RNA probes in concentrated solutions of guanidine thiocyanate.

Hybridizations with RNA probes were performed in 3-6 M guanidine thiocyanate concentrations where cells can be solubilized. At these concentrations the melting temperature of hybrids and the optimum temperature of hybrid formation are greatly reduced. The rate of hybridization in GuSCN at room temperature was greater than that in 50% formamide at 42 degrees C. Hybridizations were performed on DNA and RNA in cells dissolved in GuSCN without prior nucleic acid purification.

Animals↗