Biomedical subjects

Steven W Johnson

Publications and source records attributed to Steven W Johnson.

22 records · Page 2Linked to original sources

PubMed2002

Pharmacological identification of inward current evoked by dopamine in rat subthalamic neurons in vitro.

Dopaminergic mechanisms in the subthalamic nucleus (STN) are implicated in the pathophysiology of Parkinson's disease. Here, electrophysiological responses of STN neurons to dopamine (DA) were investigated by using whole-cell patch-clamp recordings in the rat brain slice preparation. Under current-clamp, DA depolarized membrane potential and increased the frequency of spontaneous action potentials of STN neurons. Under voltage-clamp, DA (3-300 microM) produced a reversible concentration-dependent inward current (I(DA); 6-40 pA) with an EC(50) of 13 microM. This DA-induced current had a negative slope conductance which reversed at -102 mV. It was partially reduced by barium and by superfusion with an elevated concentration of extracellular K(+). Moreover, TTX and glutamate receptor antagonists (CNQX and AP5) did not significantly affect the DA responses, indicating that I(DA) is not dependent upon afferent synaptic activity in the STN. Quinpirole, a D(2) receptor agonist, mimicked the DA action more effectively than did the D(1) agonist SKF-38393. The D(2) antagonist sulpiride, but not the D(1) antagonist SCH-23390, blocked responses induced by DA. Intracellular application of G-protein inhibitor GDP-beta-S also suppressed I(DA). GTP-gamma-S, added to the pipette solution, evoked a sustained inward shift in the absence of DA. These results suggest that DA increases the activity of STN neurons via activation of G-protein-coupled D(2)-like receptors which reduce a K(+) conductance.

Action Potentials↗

PubMed2002

Actions of orphanin FQ/nociceptin on rat ventral tegmental area neurons in vitro.

1: Non-dopamine (putative GABAergic) neurons in the ventral tegmental area are in a position to influence mesolimbic functions by their inhibitory terminals that impinge locally on dopamine neurons and via their GABAergic efferents that innervate mesolimbic structures. In the present study we investigated responses of non-dopamine and dopamine neurons, recorded intracellularly in the rat midbrain slice, to orphanin FQ/nociceptin, the endogenous ligand for opioid receptor-like orphan receptors. 2: When recording in either non-dopamine or dopamine neurons, orphanin FQ/nociceptin reduced the frequency of spike firing and caused membrane hyperpolarization under current-clamp, or produced outward current under voltage-clamp. Such responses were concentration-dependent and reversed at -108 mV and -102 mV in non-dopamine and dopamine neurons, respectively. 3: Hyperpolarizations to orphanin FQ/nociceptin were not altered by tetrodotoxin or the opioid receptor antagonist naloxone, but were reduced by the opioid receptor-like orphan receptor antagonist [Phe1(1)phiCH(2)-NH)Gly(2)]NC(1-13)NH(2) (1 microM). 4: In dopamine neurons, orphanin FQ/nociceptin reduced the frequency of bicuculline- and tetrodotoxin-sensitive spontaneous inhibitory postsynaptic potentials, and reduced the amplitude of stimulus-evoked inhibitory postsynaptic potentials. 5: Taken together, the above data provide evidence that both non-dopamine and dopamine neurons are important substrates for orphanin FQ/nociceptin within the ventral tegmental area. Simultaneous inhibition of both non-dopamine and dopamine pathways by orphanin FQ/nociceptin may account for its influences on various ventral tegmental area-related functions.

Analysis of Variance↗

PubMed2002

Decreased expression of retinol-binding proteins is associated with malignant transformation of the ovarian surface epithelium.

We have developed a modified form of suppression subtractive hybridization (SSH) that allows multiple specimens of distinct phenotypic groups to be compared for consistent differences in gene expression. We applied this system to identify genes that were expressed in normal rat ovarian surface epithelial (ROSE) cells but whose expression was lost/downregulated in four independently transformed rat ovarian cancer cell lines. Northern blot analysis using 14 of 28 nonredundant cDNA fragments from this difference library showed that the mRNA transcripts were present in normal ROSE cells but lost or markedly downregulated in four related transformed cell lines. Of particular interest, cellular retinol-binding protein 1 (CRBP1) and retinol-binding protein (RBP), two genes whose products are involved in retinol transport and metabolism, were found to be downregulated in this ovarian cancer model system. To determine if this change had relevance to human ovarian cancer, we evaluated a series of human ovarian cancer cell lines and a limited number of frozen human ovarian tumors and found lost or decreased expression of CRBP1 and RBP relative to expression in human ovarian surface epithelial (HOSE) cells. We hypothesize that the loss of CRBP1 and RBP expression disrupts retinol metabolism and retinoic acid production, which may facilitate the occurrence of genetic damage leading to the malignant transformation of the ovarian surface epithelium, the cells from which ovarian cancer arises.

Blotting, Northern↗

PubMedDate not supplied

Microarray data simulator for improved selection of differentially expressed genes.

The development of microarray technology has allowed researchers to measure expression levels of thousands of genes simultaneously. Analysis of these data requires the best normalization and statistical approaches to account for the biological and technical variability inherent in the technique. To approach this problem we have developed a publicly available simulator of microarray hybridization experiments that can be used to help assess the accuracy of bioinformatic tools in discovering significant genes. After analyzing microarray hybridization experiments from over 50 samples, an estimate of various degrees of technical and biological variability was obtained. This information was used to develop a simulator of microarray hybridization data which modeled "normal tissue samples" and "diseased tissue samples" with known, defined, changes in gene expression (a "gold standard"). The data derived from the simulator were then used to evaluate the true positive and false negative rates of several normalization procedures and gene selection techniques. We found that the type of normalization approach used was an important aspect of data analysis. Global normalization was the least accurate approach. Evaluation of gene selection techniques showed that "Significance analysis of microarrays" (SAM) and "Patterns of Gene Expression" (PaGE) were more accurate than simple t-test analysis. We provide access to the microarray hybridization simulator as a public resource for biologists to further test new emerging genomic bioinfomatic tools.

Computer Simulation↗