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L A Palmer

Publications and source records attributed to L A Palmer.

At least 19 recordsLinked to original sources
PubMed2004

Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells.

AIMS/HYPOTHESIS: Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to the reduced production of nitric oxide. The aim of this study was to investigate the regulation of endothelial nitric oxide synthase (eNOS) activity by acute and chronic elevated glucose. METHODS: Human aortic endothelial cells were cultured in 5.5 mmol/l (NG) or 25 mmol/l glucose (HG) for 4 h, 1 day, 3 days or 7 days. Mouse aortic endothelial cells were freshly isolated from C57BL/6J control and diabetic db/db mice. The expression and activity of eNOS were measured using quantitative PCR and nitrite measurements respectively. The binding of activator protein-1 (AP-1) to DNA in nuclear extracts was determined using electrophoretic mobility-shift assays. RESULTS: Acute exposure (4 h) of human aortic endothelial cells to 25 mmol/l glucose moderately increased eNOS activity and eNOS mRNA and protein expression. In contrast, chronic exposure to elevated glucose (25 mmol/l for 7 days) reduced total nitrite levels (46% reduction), levels of eNOS mRNA (46% reduction) and eNOS protein (65% reduction). In addition, AP-1 DNA binding activity was increased in chronic HG-cultured human aortic endothelial cells, and this effect was reduced by the specific inhibition of reactive oxygen species production through the mitochondrial electron transport chain. Mutation of AP-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/6J control mice, eNOS mRNA levels in diabetic db/db mouse aortic endothelial cells were reduced by 60%. This decrease was reversed by the overexpression of manganese superoxide dismutase using an adenoviral construct. CONCLUSIONS/INTERPRETATION: In diabetes, the expression and activity of eNOS is regulated through glucose-mediated mitochondrial production of reactive oxygen species and activation of the oxidative stress transcription factor AP-1.

Animals↗
PubMed2004

Contrast-dependent spatial summation in the lateral geniculate nucleus and retina of the cat.

Based on extracellular recordings from 69 lateral geniculate nucleus (LGN) cells in the anesthetized cat, we found spatial summation within their receptive fields to be dependent on the contrast of the stimuli presented. By fitting the summation curves to a difference of Gaussians model, we attributed this contrast-dependent effect to an actual change in the size of the center mechanism. Analogous changes in spatial frequency tuning were also observed, specifically increased peaks and cut-off frequencies with contrast. These effects were seen across the populations of both X and Y cell types. In a few cases, LGN cells were recorded simultaneously with one of their retinal ganglion cell (RGC) inputs (S-potentials). In every case, the RGCs exhibited similar contrast-dependent effects in the space and spatial-frequency domains. We propose that this contrast dependency in the retinal ganglion cells results directly from a reduction in the size of the center mechanism due to an increase in contrast. We also propose that these properties first arise in the retina and are transmitted passively through the LGN to visual cortex.

Action Potentials↗
PubMed2001

S-nitrosoglutathione increases cystic fibrosis transmembrane regulator maturation.

Endogenous S-nitrosoglutathione (GSNO) is known to increase the expression of certain proteins at concentrations present in the normal human airway. We hypothesized that GSNO would increase expression and maturation of the cystic fibrosis transmembrane conductance regulator (CFTR). Cells expressing DeltaF508 and wild type CFTR were exposed to GSNO and analyzed for expression and maturation by Western blot analysis. Physiologically relevant concentrations of GSNO resulted in dose- and time-dependent increases in expression. The GSNO-induced increases were eliminated by cycloheximide, suggesting a posttranscriptional effect. Unlike proteasome inhibitors, GSNO resulted in an increase CFTR maturation. The GSNO effect could be reversed by dithiothreitol and inhibited by acivicin, a gamma glutamyl transpeptidase inhibitor. These observations suggest that GSNO leads to maturation of mutated DeltaF508 CFTR, a process associated with restoration of CFTR function. Because endogenous levels of GSNO are low in the cystic fibrosis (CF) airway, these results raise the possibility that GSNO replacement therapy could be an effective treatment for CF.

Animals↗
PubMed2000

Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes.

The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia. Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1beta causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1beta showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.

Animals↗
PubMed2000

Normoxic stabilization of hypoxia-inducible factor-1 expression and activity: redox-dependent effect of nitrogen oxides.

Hypoxia-inducible factor-1 (HIF-1) is an essential transcription factor involved in the oxygen-dependent regulation of gene expression. Thiol groups in HIF-1 or in proteins that modify HIF-1 are conventional targets for regulation by nitric oxide (NO). Moreover, NO delivery to tissue by hemoglobin appears to be oxygen dependent. Therefore, the role NO plays in regulating HIF-1 activity and expression was examined. The 1-substituted diazen-1-ium-1, 2-diolate NOC-18 induced HIF-1 DNA-binding activity in normoxic bovine pulmonary artery endothelial cells and rat aortic smooth muscle cells in a time- and dose-dependent manner. Induction of HIF-1-binding activity was consistent with an increased expression of HIF-1 subunit proteins HIF-1alpha and HIF-1beta. The effect of NOC-18 on HIF-1 activity was blocked by cycloheximide, consistent with a post-transcriptional effect. NOC-18 induction of HIF-1 DNA-binding activity was not blocked with oxyhemoglobin, nor was it related to the rate of NO evolution, arguing against NO-mediation of the effect. Additionally, the effect of NOC-18 could not be mimicked by Angeli's salt, arguing against nitroxyl mediation. However, the NOC-18 effect could be reproduced by S-nitrosoglutathione (GSNO), an endogenous nitrosonium donor formed in the presence of deoxyhemoglobin. Furthermore, the GSNO effect could be reversed by dithiothreitol as well as acivicin, an inhibitor of GSNO bioactivation. Taken together, these results suggest that an S-nitrosylation reaction stabilizes HIF-1 protein expression and activity. We speculate that one signaling mechanism by which deoxyhemoglobin may activate HIF-1 involves NO.

Animals↗
PubMed1998

Hypoxia induces type II NOS gene expression in pulmonary artery endothelial cells via HIF-1.

Type II nitric oxide synthase (NOS) is upregulated in the pulmonary vasculature in a chronic hypoxia model of pulmonary hypertension. In situ hybridization analysis demonstrates that type II NOS RNA is increased in the endothelium as well as in the vascular smooth muscle in the lung. The current studies examine the role of hypoxia-inducible factor (HIF)-1 in regulating type II NOS gene expression in response to hypoxia in pulmonary artery endothelial cells. Northern blot analyses demonstrate a two fold increase in HIF-1 alpha but not in HIF-1 beta RNA with hypoxia in vivo and in vitro. Electrophoretic mobility shift assays show the induction of specific DNA binding activity when endothelial cells were subjected to hypoxia. This DNA binding complex was identified as HIF-1 using antibodies directed against HIF-1 alpha and HIF-1 beta. Transient transfection of endothelial cells resulted in a 2.7-fold increase in type II NOS promoter activity in response to hypoxia compared with nonhypoxic controls. Mutation or deletion of the HIF-1 site eliminated the response to hypoxia. These results demonstrate that HIF-1 is essential for the hypoxic regulation of type II NOS gene transcription in pulmonary endothelium.

Animals↗
PubMed1997

Regulation of major histocompatibility complex class I gene expression in thyroid cells. Role of the cAMP response element-like sequence.

The major histocompatibility complex (MHC) class I gene cAMP response element (CRE)-like site, -107 to -100 base pairs, is a critical component of a previously unrecognized silencer, -127 to -90 bp, important for thyrotropin (TSH)/cAMP-mediated repression in thyrocytes. TSH/cAMP induced-silencer activity is associated with the formation of novel complexes with the 38-base pair silencer, whose appearance requires the CRE and involves ubiquitous and thyroid-specific proteins as follows: the CRE-binding protein, a Y-box protein termed thyrotropin receptor (TSHR) suppressor element protein-1 (TSEP-1); thyroid transcription factor-1 (TTF-1); and Pax-8. TTF-1 is an enhancer of class I promoter activity; Pax-8 and TSEP-1 are suppressors. TSH/cAMP decreases TTF-1 complex formation with the silencer, thereby decreasing maximal class I expression; TSH/cAMP enhance TSEP-1 and Pax-8 complex formation in association with their repressive actions. Oligonucleotides that bind TSEP-1, not Pax-8, prevent formation of the TSH/cAMP-induced complexes associated with TSH-induced class I suppression, i.e. TSEP-1 appears to be the dominant repressor factor associated with TSH/cAMP-decreased class I activity and formation of the novel complexes. TSEP-1, TTF-1, and/or Pax-8 are involved in TSH/cAMP-induced negative regulation of the TSH receptor gene in thyrocytes, suppression of MHC class II, and up-regulation of thyroglobulin. TSH/cAMP coordinate regulation of common transcription factors may, therefore, be the basis for self-tolerance and the absence of autoimmunity in the face of TSHR-mediated increases in gene products that are important for thyroid growth and function but are able to act as autoantigens.

Animals↗
PubMed1995

Hormonal modulation of major histocompatibility complex class I gene expression involves an enhancer A-binding complex consisting of Fra-2 and the p50 subunit of NF-kappa B.

Hydrocortisone decreases major histocompatibility complex (MHC) class I gene expression in rat thyroid cells and counteracts increases induced by interferons. Using FRTL-5 cells transfected with class I promoter-reporter gene chimeras, we show that hydrocortisone action is transcriptional and mediated by an element located between 180 and 170 base pairs upstream of the start of transcription. Gel shift assays reveal that hydrocortisone causes the decrease of a specific protein-DNA complex; this same complex, referred to as Mod-1, is increased by interferon. Oligonucleotide competition assays reveal that the Mod-1 complex is associated with enhancer A of the class I gene, -180 to -170 base pairs (5'-GGGGAGTCCCC-3'), immediately upstream of the interferon response element. Antibodies to fra-2, a fos family member, and to the p50, but not the p65, subunit of NF-kappa B supershift the Mod-1 complex. We suggest that hydrocortisone decreases MHC class I gene expression by reducing the formation of Mod-1, which contains both p50 and fra-2; interferon reverses the hydrocortisone effect and increases Mod-1 formation. These observations are relevant to the molecular basis of hydrocortisone therapy in autoimmune thyroid disease and to the actions of interferon to exacerbate or induce autoimmune disease.

Animals↗
PubMed1995

Phase locking of neuronal responses to the vertical refresh of computer display monitors in cat lateral geniculate nucleus and striate cortex.

The proliferation of low-cost microcomputer systems has led to the use of these systems as alternatives to expensive display devices for visual physiology and psychophysics experiments. The video displays of these systems often lack the flexibility of achieving wide linear luminance ranges and high vertical refresh rates--two parameters which may influence data acquisition. We have examined the responses of neurons and pairs of neurons in cat LGN and striate cortex to bar and sinusoidal grating stimuli generated by a conventional PC-based VGA graphics card and displayed on a NEC Multisync + color monitor with a 60 Hz vertical (display) refresh rate. Responses to these stimuli were autocorrelated and power spectral densities (PSD) were calculated, revealing that the majority of simple and complex cortical cells and nearly all LGN cells exhibited significant peaks in their autocorrelations at 16.7 ms and in the PSD at 60 Hz. Responses to identical stimuli generated with an optical bench using an incandescent light source contained no power at 60 Hz. Furthermore, cross-correlations between the spike trains of neuron-pairs were severely contaminated by peaks directly attributable to the entrainment of the two elements of the pair to the vertical refresh signal. Thus, we suggest that the use of conventional computer displays introduces a temporal artifact into neuronal spike trains in both single and multiple spike train analysis.

Animals↗
PubMed1995

HIV Tat represses transcription through Sp1-like elements in the basal promoter.

MHC class I genes are potently repressed by HIV Tat, which transactivates the HIV LTR. Tat represses class I transcription by binding to complexes associated with a novel promoter element, consisting of Sp1-like DNA binding sites. Transcription by other Sp1-dependent promoters, such as MDR1 and the minimal SV40 promoters, is also repressed by Tat, whereas the human beta-actin promoter is neither activated by Sp1 nor repressed by Tat. Tat repression can be overcome by a strong enhancer element. Thus, the SV40 72 bp enhancer element confers protection from Tat-mediated repression on both the minimal SV40 promoter and the class I promoter. Surprisingly, Tat can activate the class I promoter in the presence of both the HIV TAR element and a strong upstream enhancer. These data demonstrate that Tat differentially affects Sp1-responsive promoters, depending on promoter architecture.

Base Sequence↗
PubMed1992

Human glucocorticoid receptor gene deletion following exposure to cancer chemotherapeutic drugs and chemical mutagens.

The sensitivity of the human glucocorticoid receptor (hGR) gene to mutagenesis by the cancer chemotherapeutic drugs adriamycin, bleomycin, and chlorambucil was evaluated using glucocorticoid-sensitive (dexs) subclones of the human leukemic cell line CEM-C7. Treatment of cells with either bleomycin or chlorambucil increased the frequency of glucocorticoid-resistant (dexr) clones 3.3- and 10-fold, respectively. Measurement of steroid-binding activity in intact dexr cells demonstrated that the predominant phenotype of drug-induced dexr clones was receptorless (r-). dexs CEM cells express only one functional hGR allele and, in addition, are heterozygous for a BclI restriction fragment length polymorphism in the hGR gene (L. A. Palmer and J. M. Harmon, Cancer Res., 51:5224-5231, 1991). To determine the basis of the r- phenotype, BclI digests of genomic DNA isolated from r+ and r- cell lines were examined for the presence of the polymorphic 2.4- and 4.4-kilobase digestion products. A deletion of all or part of the hGR gene was demonstrated by the absence of the 4.4-kilobase fragment in one of two bleomycin-induced dexr clones, as well as the ICR191-induced dexr cell line ICR27TK.3. Cytogenetic analysis of ICR27TK.3 showed that the distal portion of the long arm of one chromosome 5 had been replaced with a portion of chromosome 15. Thus, in at least two dexr cell lines, deletions and/or chromosome breaks in the hGR locus appear to account for the r- phenotype.

Alleles↗
PubMed1991

Biochemical evidence that glucocorticoid-sensitive cell lines derived from the human leukemic cell line CCRF-CEM express a normal and a mutant glucocorticoid receptor gene.

To characterize the immunoreactive glucocorticoid receptor (GR) protein present in "receptorless" (r-) mutants isolated from the glucocorticoid-sensitive (dexs) human leukemic cell line CEM-C7, binding of [3H]dexamethasone was determined in extracts prepared from the sensitive cell line 6TG1.1 and the r- mutant ICR27TK.3 after gentle freeze-thaw lysis and low-speed centrifugation. Under these conditions there was significant high-affinity binding activity in r- extracts assayed at 4 degrees C but not at 23 degrees C. Loss of binding at 23 degrees C was not a function of GR proteolysis or denaturation of the steroid-binding site and could be prevented by the addition of sodium molybdate. Dissociation of ligand from either activated or unactivated receptors in r- extracts was significantly more rapid than from receptors in extracts prepared from normal cells, suggesting that the defect in receptors in r- cells is the result of mutation in the ligand-binding site. While the rate of dissociation from unactivated receptors in r- extracts was linear, dissociation from receptors in extracts of 6TG1.1 cells was biphasic. Analysis of these dissociation curves, as well as dissociation from receptors in the B-cell line IM-9, indicated that the mutant gene present in r- cells is also present in the dexs parental cell line. This conclusion is consistent with our previous hypothesis (J.M. Harmon et al., Mol. Endocrinol., 3:734-743, 1989) that glucocorticoid-sensitive CCRF-CEM cells express both a normal (GR+) and a mutant (GR*) allele.

Binding, Competitive↗
PubMed1990

The two-dimensional spatial structure of nonlinear subunits in the receptive fields of complex cells.

We have estimated the second-order response properties of complex cells in two spatial dimensions by cross-correlating their spike trains with a binary approximation of a Gaussian white noise stimulus ensemble. Wiener-like kernels were computed and generally consisted of two or three parallel, elongated subregions alternating between augmented and suppressed response. These subunits were scattered across the receptive fields of complex cells and their axes of elongation agreed with the optimal orientation determined with drifting gratings.

Animals↗
PubMed1989

Contribution of linear spatiotemporal receptive field structure to velocity selectivity of simple cells in area 17 of cat.

We have examined the spatiotemporal structure of simple receptive fields in the cat's striate cortex by cross-correlating their spike trains with an ensemble of stimuli consisting of stationary bright and dark spots whose position was randomized on each 50 msec frame. Receptive fields were found to be either separable or inseparable in space-time and responses to moving stimuli were predicted from the spatiotemporal structure of the cell under study. Most simple cells with separable spatiotemporal receptive fields were not direction selective. All simple cells with inseparable spatiotemporal receptive fields were found to prefer movement in one direction. The optimal speed and direction were estimable from the slope of individual subregions observed in the space-time plane. The results are consistent with a linear model for direction selectivity.

Animals↗
PubMed1987

The two-dimensional spatial structure of simple receptive fields in cat striate cortex.

1. A reverse correlation (6, 8, 25, 35) method is developed that allows quantitative determination of visual receptive-field structure in two spatial dimensions. This method is applied to simple cells in the cat striate cortex. 2. It is demonstrated that the reverse correlation method yields results with several desirable properties, including convergence and reproducibility independent of modest changes in stimulus parameters. 3. In contrast to results obtained with moving stimuli, we find that the bright and dark excitatory subregions in simple receptive fields do not overlap to any great extent. This difference in results may be attributed to confounding the independent variables space and time when using moving stimuli. 4. All simple receptive fields have subregions that vary smoothly in all directions in space. There are no sharp transitions either between excitatory subregions or between subregions and the area surrounding the receptive field. 5. Simple receptive fields vary both in the number of subregions observed, in the elongation of each subregion, and in the overall elongation of the field. In contrast with results obtained using moving stimuli, we find that subregions within a given receptive field need not be the same length. 6. The hypothesis that simple receptive fields can be modeled as either even symmetric or odd symmetric about a central axis is evaluated. This hypothesis is found to be false in general. Most simple receptive fields are neither even symmetric nor odd symmetric. 7. The hypothesis that simple receptive fields can be modeled as the product of a width response profile and an orthogonal length response profile (Cartesian separability) is evaluated. This hypothesis is found to be true for only approximately 50% of the cells in our sample.

Animals↗
PubMed1987

The two-dimensional spectral structure of simple receptive fields in cat striate cortex.

1. A quantitative, general purpose method was developed for measuring the responses of visual neurons to stimuli distributed with high resolution over the two-dimensional (2D) spatial frequency domain. The stimuli consisted of drifting sinusoidal gratings of nonsaturating contrasts whose spatial frequency and orientation were drawn in random order from a 16 X 16 array of coordinates covering each neuron's responsive area. This method was applied to a population of 36 simple cells in area 17 of cat. 2. The response of each simple cell to drifting sinusoidal gratings appeared as a rectified sinusoidal modulation of the spike frequency. The degree of rectification varied from cell to cell, but for each cell, the form of the response was constant irrespective of stimulus spatial frequency, orientation, or contrast. The amplitude of the average response at the stimulus temporal frequency was used as the response metric at all spectral coordinates. Variations in this amplitude over two spectral dimensions forms a surface that we call the 2D spectral response profile. 3. For each cell, the 2D spectral response profile was localized to a limited region of the complete 2D spatial frequency domain. In bidirectionally responsive cells, there were two lobes in the surface disposed with mirror symmetry about the origin. In all cells, each lobe exhibited a single maximum and the response decayed smoothly in every direction away from the maximum. Isoresponse amplitude contours were elliptical and often, but not always, elongated about an axis of symmetry passing through the origin. 4. We tested the hypothesis that orientation and spatial frequency tuning are independent by forming scaled radial and angular sections through 2D spectral response profiles. In virtually every case polar separability did not obtain, that is, orientation selectivity depended on spatial frequency and vice versa. 5. In contrast, more than half the cells had 2D spectral response profiles that were Cartesian separable. The 2D spectral response profiles of most of the remaining cells were neither polar nor Cartesian separable, because the response profiles were elongated about an axis of symmetry that did not pass through the origin. 6. These results are discussed in terms of the constraints they place on models of the contributions simple cells make toward the neural representation of images.

Animals↗
PubMed1987

An evaluation of the two-dimensional Gabor filter model of simple receptive fields in cat striate cortex.

1. Using the two-dimensional (2D) spatial and spectral response profiles described in the previous two reports, we test Daugman's generalization of Marcelja's hypothesis that simple receptive fields belong to a class of linear spatial filters analogous to those described by Gabor and referred to here as 2D Gabor filters. 2. In the space domain, we found 2D Gabor filters that fit the 2D spatial response profile of each simple cell in the least-squared error sense (with a simplex algorithm), and we show that the residual error is devoid of spatial structure and statistically indistinguishable from random error. 3. Although a rigorous statistical approach was not possible with our spectral data, we also found a Gabor function that fit the 2D spectral response profile of each simple cell and observed that the residual errors are everywhere small and unstructured. 4. As an assay of spatial linearity in two dimensions, on which the applicability of Gabor theory is dependent, we compare the filter parameters estimated from the independent 2D spatial and spectral measurements described above. Estimates of most parameters from the two domains are highly correlated, indicating that assumptions about spatial linearity are valid. 5. Finally, we show that the functional form of the 2D Gabor filter provides a concise mathematical expression, which incorporates the important spatial characteristics of simple receptive fields demonstrated in the previous two reports. Prominent here are 1) Cartesian separable spatial response profiles, 2) spatial receptive fields with staggered subregion placement, 3) Cartesian separable spectral response profiles, 4) spectral response profiles with axes of symmetry not including the origin, and 5) the uniform distribution of spatial phase angles. 6. We conclude that the Gabor function provides a useful and reasonably accurate description of most spatial aspects of simple receptive fields. Thus it seems that an optimal strategy has evolved for sampling images simultaneously in the 2D spatial and spatial frequency domains.

Animals↗