Search PubMed⌕ Search

Biomedical subjects

C S Watson

Publications and source records attributed to C S Watson.

At least 37 records · Page 2Linked to original sources
PubMed1999

Estrogen receptor-alpha detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry.

A population of estrogen receptor-alpha (ER alpha) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ER alpha at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2% paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ER alpha. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ER alpha Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity of staining for ER alpha was confirmed by three approaches: first, treatment with an antisense oligodeoxynucleotide to nuclear ER alpha mRNA reduced immunolabeling of both membrane and nuclear ER alpha; second, labeling by two Abs raised against different ER alpha oligopeptides was neutralized by competing peptide; third, six Abs (ER21, H226, R4, H222, MC20, and C542) that recognize unique epitopes on rodent ER alpha produced immunolabeling, but neither primate-specific ER alpha Ab nor Ab to ER beta caused staining. In addition to demonstrating the plasma membrane ER alpha in GH3/B6/F10 cells, this method should be applicable to other cell types that exhibit nongenomic responses to estrogen or other steroid hormones.

Aldehydes↗
PubMed1998

Prostaglandin E2 inhibition of fetal breathing movements is not sustained during prolonged reduced uterine blood flow in sheep.

Fetal breathing movements (FBM) are inhibited by both exogenous prostaglandin E2 (PGE2) and ethanol in sheep. Maternal ethanol exposure in late-gestation sheep also increases fetal [PGE2]. However, during prolonged reduced uterine blood flow (RUBF) when [PGE2] in fetal plasma is already elevated, FBM are not inhibited by ethanol. These experiments were designed, therefore, to test the hypothesis that the FBM response to PGE2 is also diminished during RUBF. PGE2 (594+/-19 ng.min(-1).kg(-1) fetal body weight) was infused for 6 h into the jugular vein of RUBF (PO2 = 14+/-1 mmHg (1 mmHg = 133.3 Pa); n = 7) and control (PO2 = 22+/-1 mmHg (p < 0.01); n = 7) ovine fetuses, and the effect on FBM, electrocortical (ECoG), and electroocular activities was determined. The infusion of PGE2 increased plasma [PGE2] from 881+/-162 to 1189+/-114 pg.mL(-1) in RUBF fetuses and from 334+/-72 to 616+/-118 pg.mL(-1) (p < 0.05) in control fetuses. FBM were initially inhibited by PGE2 from 22.5+/-9.4 and 17.9+/-6.5% of the time to 6.9+/-2.4 and 0.5+/-0.4% (p < 0.01) in RUBF and control fetuses, respectively. FBM remained inhibited in control fetuses throughout the infusion but returned to baseline incidence in RUBF fetuses in the last 2 h of the infusion. These results are consistent with the hypothesis that one component of the adaptative mechanisms of the fetus to prolonged RUBF is an altered response of FBM to exogenous PGE2. We speculate that the lack of a sustained inhibition in FBM during RUBF with infusion of PGE2 may be a result of an alteration in brainstem receptor function or number or local PGE2 removal.

Animals↗
PubMed1997

Cell cycle regulation of membrane glucocorticoid receptor in CCRF-CEM human ALL cells: correlation to apoptosis.

The human leukemic cell line (CCRF-CEM) and a subline enriched for the plasma membrane-resident glucocorticoid receptor (mGR) were studied for the influence of the cell cycle on the expression and function of mGR. Three synchronization procedures (double thymidine, colcemid, and combined thymidine-colcemid blocks) were used. Fluorescent microscopy and flow cytometry simultaneously assessed antibody-tagged mGR and DNA. In addition, mGR was quantitated and characterized by immunoprecipitation and immunoblotting. Apoptosis was assayed by DNA fragmentation (TUNEL assay) and by cell survival (trypan blue exclusion). All synchronization procedures demonstrated that progression from DNA replication (S) to the second growth phase and mitosis (G2/M) leads to cells having the highest levels of mGR expression and being highly glucocorticoid sensitive in the apoptosis assays: 32 and 80% sensitivity of wild type and mGR-enriched cells, respectively, compared with 12 and 30% sensitivity in asynchronous cells. Therefore, mGR expression appears to be cell cycle regulated, with its highest expression at late S-G2/M, when the cells are most sensitive to the lymphocytolytic effects of glucocorticoids.

Apoptosis↗
PubMed1996

Detection of frequency changes in transposed sequences of tones.

The ability to detect frequency changes in transposed sequences of tones was examined in a series of seven experiments. Listeners were asked to judge which of two transposed (i.e., frequency-shifted) comparison patterns preserved the sequence of relative frequencies presented in a preceding standard pattern. The task was performed with five-tone and two-tone patterns under conditions of high and minimal pattern uncertainty. Regardless of pattern length or level of uncertainty, frequency discrimination thresholds for a change in the relative frequency of a single tone were considerably higher when patterns were transposed than when they were not. There was a tendency for performance to worsen with increasing degrees of transposition (primarily under high uncertainty) but most of the detrimental effects of transposition occurred within the first two semitones of transposition. Minimal uncertainty testing resulted in large improvements with five-tone patterns (as much as one order of magnitude), but there was no effect of level of uncertainty on performance with two-tone patterns. Thresholds for changes in two-tone patterns were similar to (although slightly higher than) those for five-tone patterns under minimal-uncertainty testing. This pattern of results reveals that the effects of stimulus complexity (sequence length) and pattern familiarity (level of uncertainty) on relative-frequency discrimination are quite similar to the effects of these variables on absolute-frequency discrimination.

Auditory Perception↗
PubMed1996

Auditory and visual speech perception: confirmation of a modality-independent source of individual differences in speech recognition.

Two experiments were run to determine whether individual differences in auditory speech-recognition abilities are significantly correlated with those for speech reading (lipreading), employing a total sample of 90 normal-hearing college students. Tests include single words and sentences, recorded on a videodisc by a male speaker [Bernstein and Eberhardt, Johns Hopkins Lipreading Corpus, The Johns Hopkins University, Baltimore, MD, 1986]. The auditory speech was presented with a white noise masker, at -7 dB Sp/N. The correlations between overall auditory and visual performance were 0.52 and 0.43 in the two experiments, consistent with the existence of a modality-independent ability to perceive linguistic "wholes" on the basis of linguistic fragments. Subjects in the second experiment also identified printed sentences, with 40%-60% portions of the printed characters deleted. Performance on this graphical "fragmented-sentences test" also correlated significantly with auditory speech recognition, providing a possible clue to the cognitive basis for the look-versus-listen correlation. The existence of a modality-independent source of variance in speech-recognition abilities may be a partial explanation of the difficulty in demonstrating strong associations between psychoacoustic measures of spectral or temporal acuity, and speech discrimination or identification. Female subjects in both experiments were significantly better lipreaders than their male counterparts.

Adolescent↗
PubMed1996

Auditory psychophysics and perception.

In this review of auditory psychophysics and perception, we cite some important books, research monographs, and research summaries from the past decade. Within auditory psychophysics, we have singled out some topics of current importance: Cross-Spectral Processing, Timbre and Pitch, and Methodological Developments. Complex sounds and complex listening tasks have been the subject of new studies in auditory perception. We review especially work that concerns auditory pattern perception, with emphasis on temporal aspects of the patterns and on patterns that do not depend on the cognitive structures often involved in the perception of speech and music. Finally, we comment on some aspects of individual difference that are sufficiently important to question the goal of characterizing auditory properties of the typical, average, adult listener. Among the important factors that give rise to these individual differences are those involved in selective processing and attention.

Adult↗
PubMed1996

Fetal breathing is not inhibited by ethanol exposure during prolonged reduced uterine blood flow.

When prolonged hypoxemia is induced in fetal sheep by reducing uterine blood flow, fetal breathing movements (FBM) return to normal incidence after their initial decrease. Ethanol also inhibits FBM. These experiments were designed to determine the role of fetal oxygenation status in affecting ethanol-induced inhibition of FBM. A 1-h maternal infusion of 1 g ethanol/kg maternal body weight was given to animals following 20 h of reduced uterine blood flow (RUBF; n = 9), as well as to normoxemic (n = 9) fetal sheep, and the effect on FBM, electrocortical activity (ECoG), and electroocular activity (EOG) was determined. In normoxemic fetuses, the incidence of FBM decreased from 26.1 +/- 5.5% to 2.8 +/- 1.6% (p < 0.05) with ethanol, subsequently returning to baseline values over 6 h, but there was no effect of ethanol on FBM incidence in RUBF fetuses (32.8 +/- 9.1%). Ethanol increased the incidence of intermediate ECoG from 16.6 +/- 2.9% to 49.0 +/- 9.8% (p < 0.01) in normoxemic fetuses, but there was no change in RUBF fetuses. We conclude that the adaptive mechanism(s) invoked by fetal sheep during prolonged RUBF abolishes its ability to respond behaviourally to ethanol.

Animals↗
PubMed1995

Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding.

GH3/B6 rat pituitary tumor cells exhibit rapid prolactin release (within 5 min) when treated with nanomolar amounts of estrogen. However, the putative protein mediator of this nongenomic action has not been described. Using antibodies directed against a peptide representing the hinge region of the intracellular estrogen receptor (iER), we have demonstrated that these cells contain a membrane ER (mER). We now report that confocal scanning laser microscopy of cells labeled live with the anti-peptide antibody further supports a membrane localization of ER. The monoclonal antibodies H226 and H222 and a polyclonal antibody, ER21, each recognizing a unique epitope on iER (NH2 terminal to the DNA-binding region, within the steroid binding region, and the NH2-terminal end, respectively), also immunohistochemically label membrane proteins of immuno-selected GH3/B6 cells. These cells also specifically bind a fluorescent estrogen-BSA conjugate. Coincubation of cells with anti-ER antibody and the fluorescent estrogen-BSA conjugate reveals that these labels colocalize on cells. These results suggest that mER may be structurally similar to iER.

Animals↗
PubMed1995

The other estrogen receptor in the plasma membrane: implications for the actions of environmental estrogens.

Environmental or nutritional estrogenic toxicants are thought to mediate developmental and carcinogenic pathologies. Estrogen receptor (ER) measurements are currently used to predict hormonal responsiveness; therefore all ER subpopulations should be considered. We have been involved in the immunoidentification and characterization of membrane steroid receptors in several systems and have recently shown that binding of estradiol (E2) to a subpopulation of ERs (mER) residing in the plasma membrane of GH3 pituitary tumor cells mediates the rapid release of prolactin (PRL). Here we review these findings and present other important characterizations of these receptors such as trypsin and serum susceptibility, movement in the membrane, confocal localization to the membrane, binding to and function of impeded ligands, and immunoseparation of cells bearing mER. We plan to use this system as a model for both the physiological and pathological nongenomic effects of estrogens and estrogenic xenobiotics. Specifically, it should be useful as an in vitro assay system for the ability of estrogenic xenobiotics to cause rapid PRL release as an example of nongenomic estrogen effects.

Cell Membrane↗
PubMed1994

Identification of vitamin D-stimulated alkaline phosphatase in IEC-6 cells, a rat small intestine crypt cell line.

The IEC-6 cell line was derived from newborn rat small intestinal crypts and maintains characteristics of nontransformed crypt epithelial cells. We have reported that alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated in IEC-6 cells by 1,25-dihydroxycholecalciferol (1,25(OH)2D3). Using reverse transcription polymerase chain reaction, we determined that the ALP in IEC-6 cells is the liver/bone/kidney ALP (LALP) isoenzyme and not the mature enterocyte form. Levamisole, a specific blocker for LALP, blocked the ALP activity of IEC-6 cells. Kinetic studies showed that 1,25(OH)2D3 increases Vm values of ALP in IEC-6 cells while Km values remain the same. Northern analysis of LALP mRNA levels in IEC-6 cells indicated that 10(-7) M 1,25(OH)2D3 acts by increasing alkaline phosphatase activity at the gene expression level.

Alkaline Phosphatase↗
PubMed1994

Formant-frequency discrimination for isolated English vowels.

Thresholds for formant-frequency discrimination were obtained for ten synthetic English vowels patterned after a female talker. To estimate the resolution of the auditory system for these stimuli, thresholds were measured using well-trained subjects under minimal-stimulus-uncertainty procedures. Thresholds were estimated for both increments and decrements in formant frequency for the first and second formants. Reliable measurements of threshold were obtained for most formants tested, the exceptions occurring when a harmonic of the fundamental was aligned with the center frequency of the test formant. In these cases, unusually high thresholds were obtained from some subjects and asymmetrical thresholds were measured for increments versus decrements in formant frequency. Excluding those cases, thresholds for formant frequency, delta F, are best described as a piecewise-linear function of frequency which is constant at about 14 Hz in the F1 frequency region (< 800 Hz), and increases linearly in the F2 region. In the F2 region, the resolution for formant frequency is approximately 1.5%. The present thresholds are similar to previous estimates in the F1 region, but about a factor of three lower than those in the F2 region. Comparisons of these results to those for pure tones and for complex, nonspeech stimuli are discussed.

Adult↗
PubMed1994

Effects of decision criterion on response latencies of binary decisions.

The effects of decision criterion on response latencies of binary decisions were examined. The stimuli comprised two, partly overlapping, "normal" distributions of either two-digit numbers or tonal frequencies. Individual stimuli were randomly sampled from the distributions, and subjects had to decide from which distribution the stimulus was sampled. The decision goal was to maximize the expected gain using three different payoff matrices. Decision latencies with and without prior knowledge of optimal decision criteria were measured. In a control task, subjects were asked only to label stimuli as being either above or below the optimal criteria (without regard for a posteriori probabilities or expected gain). The relation between criterion and latency was examined by means of a trial-by-trial analysis of the stimuli and the responses. Response latency was inversely related both to the distance between the stimulus and the decision criterion and to the probability of the response elicited by the stimulus. Response latencies showed a maximum at the criterion in all conditions. These results were obtained with different stimulus modalities (tones or numbers), different discriminability levels within the auditory modality, and different decision tasks. It is proposed that the decision criterion is the primary determinant of response latencies of binary decisions.

Acoustic Stimulation↗
PubMed1993

Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients.

Anti-peptide antibody to the human glucocorticoid receptor (GR) was produced and used to demonstrate that a subset of the GR population resides in the plasma membrane of human leukemic cells. Characterization of the antibody with intracellular GR (iGR) showed its ability to shift [3H]triamcinolone acetonide-labeled GR (4S protein) from two human leukemic cell lines to a higher density in sucrose gradients; Western and autoradiographic analysis of affinity-labeled ([3H]dexamethasone 21-mesylate) receptor revealed an immunoreactive and competitively labeled band of 94 kDa. CCRF-CEM cell membrane GR (mGR) resolved as a > 7S protein on density gradients and immunoselected cell surface protein labeled by whole cell biotinylation or affinity-labeling with [3H]dexamethasone 21-mesylate was approximately 145 kDa, demonstrating that mGR was larger in size than iGR, as has been shown previously for the mGR of mouse lymphoma cells. Analysis of mGR in lymphocytes of leukemic patients and the CCRF-CEM cell line indicated differences in levels of expression as shown by FACS and immunocytochemical analyses. We are currently using this system to study the correlation between the quantity of membrane-resident GRs and the glucocorticoid-induced lytic response, a relationship previously shown in the murine (S-49 cell) system.

Amino Acid Sequence↗
PubMed1993

The presence of functional estrogen receptors in intestinal epithelial cells.

Reproductive and maturational nutritive needs are examples of situations in which alterations in circulating concentrations of estrogens are associated with changes in intestinal epithelial function. However, it is not clear that any of these effects is due to direct interaction of estrogen with intestinal epithelial estrogen receptors (ER). The experiments reported here were designed to determine whether the small intestinal epithelium contains functional ER and might, therefore, be an estrogen-responsive tissue. IEC-6 cells, a non-transformed line of cells isolated from rat small intestinal crypts, were used for many of the experiments, because they provide a pure preparation of crypt epithelial cells. IEC-6 cells were found to exhibit specific saturable binding of estradiol with a Kd of 5 x 10(-10) M and approximately 100 binding sites/cell. Reverse transcriptase-polymerase chain reaction demonstrated that IEC-6 cells as well as epithelial cells from each segment of the rat intestine (duodenum, jejunum, ileum, and colon) contained ER mRNA of the sequence determined from rat uterus. Estradiol was shown to stimulate IEC-6 cell c-fos mRNA content rapidly and transiently in a manner analogous to that which has been previously demonstrated for other estrogen-responsive tissues. These data demonstrate that intestinal epithelial cells contain ER capable of regulating gene transcription and provide the basis for future studies designed to elucidate the role of estrogens in the regulation of intestinal epithelial function and pathophysiology.

Animals↗
PubMed1992

The "proportion-of-the-total-duration rule" for the discrimination of auditory patterns.

A principle of auditory perception that governs the detectability of changes in components in unfamiliar sequences of tones is demonstrated in four experiments. The proportion-of-the-total-duration (PTD) rule can be stated as follows: Each individual component of an unfamiliar sequence of tones is resolved with an accuracy that is a function of its proportion of the total duration of the sequence or "pattern." An adaptive-tracking frequency-discrimination task was used in all experiments. Experiment 1 demonstrated that the PTD rule holds over a wide range of total pattern durations, numbers of components, and component durations. Experiment 2 demonstrated that the PTD rule governs discrimination performance despite variation in the relative durations of context and target tones. Experiment 3, using a variable temporal position for the target, confirmed that the PTD effect does not require that a listener be able to anticipate the temporal location of the target tone. Experiment 4, using two target tones, showed that the PTD rules applies to the proportional duration of individual components within patterns and not to the total proportional duration of nonadjacent components within the pattern. These findings are incompatible with performance limitations based on a fixed-duration short-term memory capacity and with versions of informational limitations in which the amount of information in a pattern varies either with the number of components or with the total pattern duration. The PTD rule appears to reflect the way listeners distribute their attention when presented with unfamiliar complex sounds that have no structural properties (other than proportional duration) that significantly increase the salience of individual components.

Acoustic Stimulation↗