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Biomedical subjects

D A Wilkinson

Publications and source records attributed to D A Wilkinson.

At least 19 recordsLinked to original sources
PubMed1992

A human endogenous long terminal repeat provides a polyadenylation signal to a novel, alternatively spliced transcript in normal placenta.

We have been investigating the impact that the long terminal repeats (LTRs) of the RTVL-H family of human endogenous retroviral-like elements may have on the expression of adjacent cellular genes. Using a differential hybridization strategy, we have screened a cDNA library from a normal full-term human placenta and have identified two clones containing non-RTVL-H-related cellular sequences that have been polyadenylated within an RTVL-H LTR. One of these clones, cPj-LTR, contains an open reading frame (ORF) of 223 amino acids. Southern analysis indicated that the corresponding gene, termed PLT, is most probably a single multi-exon locus and that related sequences are present in the mouse genome, suggesting that this gene has been evolutionarily conserved. Database searches detected no significant homology to previously published sequences, indicating that PLT is a novel gene. Northern analysis identified several PLT-related transcripts in placental RNA samples, one of which is associated with the LTR. The presence of this PLT-LTR fusion transcript in normal placenta was also confirmed by PCR. Additional hybridization studies with RNAs from various cell lines suggested that the PLT locus is differentially expressed in different cell types. To investigate the structure of the non-LTR-associated PLT-related transcripts, additional clones were isolated from the placental cDNA library. Analysis of these clones suggests that the PLT mRNA undergoes alternative splicing at its 3' end, with polyadenylation within an RTVL-H LTR occurring in one of the resulting transcripts.

Alternative Splicing↗
PubMed1992

A.T.L.S. on the ski slopes--a steamboat experience.

The Steamboat Mountain Ski Patrol incorporates local emergency physicians and a visiting trauma surgeon as a second-tier response to life-threatening mountain events. The case of a 48-year-old man surviving a potentially lethal postinjury tension pneumothorax and a review of the 1989-1990 major trauma experience on this ski mountain underscores the value of this concept.

Adolescent↗
PubMed1992

M2 muscarinic ([3H]N-methyl scopolamine) binding in micropunches of rat ventricular myocardium: characterization and modification by progesterone.

A new technique is outlined for the characterization and quantification of M2 muscarinic binding sites (receptors) in micropunches (1 mm diam.), cut from slices (350 microns), of fresh cardiac tissue using the hydrophilic antagonist [3H]N-methyl scopolamine. The use of this water-soluble ligand allows us to label, and quantify, M2 receptors on the cell surface of intact cells contained within the micropunch. We believe that cardiac micropunches offer a simple but powerful approach to the investigation of membrane receptor regulation in tissue that largely retains the in vivo cytoarchitecture. Specific binding is reversible, stereospecific, saturable, of high affinity, and has the drug specificity typical of an M2 muscarinic receptor. In rat left ventricle, Bmax was 151.2 +/- 10.3 fmol/mg protein while KD was 1.0 +/- 0.1 nM. Nonspecific binding of the ligand was very low, varying from 2.8% (at 0.27 nM) to 7.7% (at 3.58 nM). This micropunch assay was used to determine that progesterone can compete with the muscarinic ligand for the M2 receptor in vitro (IC50 = 50 x 10(-6) M). The steroids estradiol and testosterone, as well as ouabain, were without effect. Progesterone inhibited [3H]N-methyl scopolamine binding competitively (KD reduced from 1.9 to 4.3 nM) without affecting the rate of association of the ligand. However, progesterone induced a rapid dissociation of the ligand from its receptor. We conclude that the micropunch assay described here is suitable for the continued study of sex hormone effects on cardiac function.

Animals↗
PubMed1991

Reversible effects of acute and long-term administration of delta-9-tetrahydrocannabinol (THC) on memory in the rat.

A study was designed to develop a measure of both acute and chronic effects of THC administration on memory in the rat. Errors in an 8-arm radial maze, before and after two delay intervals (5 s and 1 h, introduced between the fourth and the fifth arm choice), constituted the principal dependent measures. The first experiment involved testing the animals shortly after administration of 1.25 mg/kg THC. The drug did not affect performance in the pre-delay tests, although a significant effect was observed after the 5-s delay but not after 1-h delay. In the second experiment, 5 mg/kg THC or saline were administered 6 days/week for 90 days. Testing was conducted 18 h after each drug administration. During chronic administration the pre-delay performance did not differ between groups but the post-delay performance of the THC group deteriorated in a gradual manner, relative to their controls, in both the 5-s and 1-h delay conditions. After discontinuation of drug administration, the differences between groups reversed only after 30 days. The results provided evidence that both acute and chronic administration of THC affected working-memory in the radial arm maze test, although it did not interfere with the general cues of the task (reference memory). Chronic drug effects on memory were reversible after prolonged abstinence. Thus, the 8-arm radial maze task proved to be a useful measure of THC effects on memory and could be further used to investigate more thoroughly the mechanisms involved in such drug effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Routine culturing of normal, dysplastic and malignant human mammary epithelial cells from small tissue samples.

We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca++ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca++ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely produce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression.

Adult↗
PubMed1990

Autonomous expression of RTVL-H endogenous retroviruslike elements in human cells.

Northern (RNA) blot analysis of RNA from various human cell lines and tissues has demonstrated that elements belonging to the RTVL-H family of human endogenous retroviruslike sequences are expressed in several cell types. The highest levels of RTVL-H-related RNAs were observed in teratocarcinoma cell line NTera2D1, HeLa cells, two bladder carcinoma cell lines, and normal amniotic tissue. Expression was also observed in normal chorion and in some other cell lines. The RTVL-H transcription pattern varied among the different cell types, but several expressed a unit-length 5.6-kilobase transcript. Characterization of cDNA clones corresponding to transcripts present in NTera2D1 cells indicates that the complex transcription pattern observed in these cells is generated by the following: (i) transcription of both full-length and deleted genomic elements, which is initiated within the 5' long terminal repeat (LTR) and, in all but one case, polyadenylated in the 3' LTR; (ii) the splicing of both unit-length transcripts and transcripts from a deleted element; (iii) transcription involving solo LTR sequences; and (iv) transcription which, in one case, reads through the 3' LTR into flanking cellular sequences. Sequence data obtained from 25 cDNA clones revealed that at least 13 RTVL-H elements are expressed in NTera2D1 cells. The positions of several termination codons within the pol region are the same among nine different elements, indicating that an ancestral RTVL-H element bearing these mutations dispersed within the genome. We also found that RTVL-H expression varied among samples of amnion and chorion tissue isolated from different individuals. These findings demonstrate that regulated autonomous expression of RTVL-H sequences occurs in human cells.

Amino Acid Sequence↗
PubMed1989

Comparing tobacco cigarette dependence with other drug dependencies. Greater or equal 'difficulty quitting' and 'urges to use,' but less 'pleasure' from cigarettes.

About 1000 persons seeking treatment for alcohol or drug dependence were asked, relative to cigarettes, about the difficulty of quitting the use of the substance for which they were seeking treatment, the strength of their strongest urges to use, and the pleasure they derived from use. Fifty-seven percent said that cigarettes would be harder to quit using than their problem substance. These ratings were related to the level of cigarette dependence and the perceived difficulty of quitting the use of the problem substance. The alcohol-dependent persons were about four times more likely than the drug-dependent persons to say that their strongest urges for cigarettes were at least as great as their strongest urges for their problem substance. Cigarettes were generally rated as less pleasurable than alcohol or other drugs. Thus, experiential experts on dependence judge cigarette dependence as at least as "addictive" as other drug use, but not as pleasurable, indicating important similarities and differences between cigarette dependence and other forms of dependence on psychoactive substances.

Adult↗
PubMed1989

"Cravings" are ambiguous: ask about urges or desires.

Despite the dictionary definition of "craving" (a strong desire), two studies indicate that a substantial percentage of persons with alcohol and drug problems use the word "craving" to mean any desire or urge, even a weak one, to use substances. Researchers and clinicians are advised to beware of this ambiguity of "craving" and to consider the conceptual status of "craving" in their work.

Adult↗
PubMed1988

Specific volumes of lipids in fully hydrated bilayer dispersions.

The neutral buoyancy method of obtaining absolute specific volumes of lipid in multilamellar dispersions is critically investigated. Control experiments show that there is no preferential partitioning of 2H2O vs. H2O into the liposomes, and several thermodynamic properties of the samples, such as the enthalpy change and the volume change of the main transition, are changed very little with deuteration of the solvent. The assumption that the molecular volume of the solvent in the interlamellar space is essentially the same as in bulk solution is discussed; and it is shown to introduce rather small corrections. Previous procedures have been modified to avoid possible kinetic limitations in phases with low water permeability. It is concluded that the molecular volume of lipid in bilayers can be obtained to an accuracy better than 0.002 nm3 (2A3) which is less than 0.2% of typical molecular volumes of lipids.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1988

Validation of self-reported cannabis use by urine analysis.

A study of 113 young multiple drug users, interviewed at one year follow-up, was designed to assess the validity of self-reported cannabis use. Subjects provided information about the recency, frequency and typical dose of their use of cannabis in the preceding 30 days. Urine samples were collected and analyzed for delta 9 THC metabolites. Recency of use was the best predictor of urine test result, but a measure of frequency of use significantly increased the proportion of variance accounted for. The results strongly supported the validity of self-reported cannabis use and indicate that valid self-reports of drug consumption can be obtained from multiple drug users in treatment.

Adult↗
PubMed1988

Theoretical aspects of stripe formation in relation to Drosophila segmentation.

Many aspects of Drosophila segmentation can be discussed in one-dimensional terms as a linear pattern of repeated elements or cell states. But the initial metameric pattern seen in the expression of pair-rule genes is fully two-dimensional, i.e. a pattern of stripes. Several lines of evidence suggest a kinetic mechanism acting globally during the syncytial blastoderm stage may be responsible for generating this pattern. The requirement that the mechanism should produce stripes, not spots or some other periodic pattern, imposes preconditions on this act, namely (1) sharp anterior and posterior boundaries that delimit the pattern-forming region, and (2) an axial asymmetrizing influence in the form of an anteroposterior gradient. Models for Drosophila segmentation generally rely on the gradient to provide positional information in the form of concentration thresholds that cue downstream elements of a hierarchical control system. This imposes restrictions on how such models cope with experimental disturbances to the gradient. A shallower gradient, for example, means fewer pattern elements. This need not be the case if the gradient acts through a kinetic mechanism like reaction-diffusion that involves the whole system. It is then the overall direction of the gradient that is important rather than specific concentration values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Tris buffer causes acyl chain interdigitation in phosphatidylglycerol.

The structure of the gel phase and the properties of the acyl chain disordering transition of dipalmitoyl phosphatidylglycerol (DPPG) have been studied using differential scanning calorimetry, differential scanning dilatometry, and X-ray diffraction. In the presence of small, monovalent cations, DPPG at 22 degrees C exists in a lamellar phase in which the hydrocarbon chains are tilted from the perpendicular to the bilayer surface. Around 34 degrees C, there is a small pretransition (delta H less than 1 kcal/mol) followed by the main transition at 40.4 degrees C (delta H = 8.3 kcal/mol; delta V = 0.0381 ml/g). If DPPG is suspended in Tris-HCl buffer in the absence of other monovalent cations, X-ray diffraction data show that at 22 degrees C, the gel phase consists of interdigitated acyl chains perpendicular to the plane of the bilayer. No pretransition is observed and the main transition occurs at 41.3 degrees C with delta H = 9.1 kcal/mol and delta V = 0.0514 ml/g. If sufficient Na+ or K+ ions are added to the Tris-buffered DPPG, the phase behavior reverts to what is observed in the absence of Tris. Analysis of the energetics of the main transition shows that the increase in van der Waals interaction energy resulting from the larger delta V in Tris can be compensated by the favorable energetics of removing terminal methyl groups from the bilayer surface. The amount of disordering, i.e. formation of gauche rotamers, is likely to be the same in Tris as it is in buffers without amphiphilic cations.

Calorimetry, Differential Scanning↗
PubMed1987

Reversibility of alcohol-related brain damage: clinical and experimental observations.

Chronic alcoholics who maintain abstinence often demonstrate remarkable improvement of neurological and mental dysfunction. This paper presents an overview of the clinical and laboratory work of our group. Reversible clinical manifestations include psychometric scores, ataxia, tremor, Parkinsonism, dyskinesia, cerebral atrophy, EEG parameters, and a CSF acidosis. Electrophysiological investigations showed that in the in vitro hippocampus of rats fed ethanol for several months there was evidence for diminished long-term potentiation, impaired neuronal inhibitory mechanisms (diminished inhibitory post-synaptic potentials and post-spike after hyperpolarisations), decreased neuronal specific membrane capacitance and increased specific membrane resistance. Golgi stains showed attenuation of hippocampal CA1 neuronal dendrites in rats fed ethanol for five months, which reverted to control size in rats permitted two months of alcohol withdrawal.

Action Potentials↗
PubMed1987

The chronic effects of alcohol on memory. A contrast between a unitary and dual system approach.

Recent studies have indicated that impairment of memory is a common cognitive deficit related to long-term alcohol consumption. Such deficits range from subtle disturbance through a "subclinical" amnesic disorder to full-blown Korsakoff syndrome. This finding is congenial with the hypothesized continuum of alcohol-related impairment of memory. Among alcoholics, this memory impairment appears to be etiologically distinct from the commonly observed deficits in abstracting and problem-solving abilities. In the present chapter, a dual and a unitary formulation of memory are contrasted in their ability to present a coherent account of what memory tasks Korsakoff amnesics can and cannot do. The explanations offered by unitary theory are weak compared to those derived from the theory that memory consists of two distinct systems--experiential and abstractive memory. We indicate how the pattern of responding on standard clinical tests of memory can be coherently analyzed post hoc within the dual formulation. A number of experimental predictions are also presented based on the theory that Korsakoff amnesics have a profound impairment of experiential memory, whereas the abstractive system is essentially normal. Such experiments should clarify the nature both of Korsakoff amnesia and of memory itself. In conclusion, we indicated that memory disorders related to alcoholism can be understood in terms of a gradient, or continuum, of impairment of the experiential system.

Alcohol Amnestic Disorder↗