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J W Miller

Publications and source records attributed to J W Miller.

At least 55 records · Page 3Linked to original sources
PubMed1999

Visualization of double-stranded RNAs from the myotonic dystrophy protein kinase gene and interactions with CUG-binding protein.

Myotonic dystrophy (DM) is associated with a (CTG) (n) triplet repeat expansion in the 3'-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Using electron microscopy, we visualized large RNAs containing up to 130 CUG repeats and studied the binding of purified CUG-binding protein (CUG-BP) to these RNAs. Electron microscopic examination revealed perfect double-stranded (ds)RNA segments whose lengths were that expected for duplex RNA. The RNA dominant mutation model for DM pathogenesis predicts that the expansion mutation acts at the RNA level by forming long dsRNAs that sequester certain RNA-binding proteins. To test this model, we examined the subcellular distribution and RNA-binding properties of CUG-BP. While previous studies have demonstrated that mutant DMPK transcripts accumu-late in nuclear foci, the localization pattern of CUG-BP in both normal and DM cells was similar. Although CUG-BP in nuclear extracts preferentially photocrosslinked to DMPK transcripts, this binding was not proportional to (CUG) (n) repeat size. Moreover, CUG-BP localized to the base of the RNA hairpin and not along the stem, as visualized by electron micro-scopy. These results provide the first visual evidence that the DM expansion forms an RNA hairpin structure and suggest that CUG-BP is unlikely to be a sequestered factor.

3' Untranslated Regions↗
PubMed1999

L-proline and L-pipecolate induce enkephalin-sensitive currents in human embryonic kidney 293 cells transfected with the high-affinity mammalian brain L-proline transporter.

The high-affinity mammalian brain L-proline transporter (PROT) belongs to the GAT1 gene family, which includes Na- and Cl-dependent plasma membrane carriers for neurotransmitters, osmolites, and metabolites. These transporters couple substrate flux to transmembrane electrochemical gradients, particularly the Na gradient. In the nervous system, transporters clear synapses and help to replenish transmitters in nerve terminals. The localization of PROT to specific excitatory terminals in rat forebrain suggests a role for this carrier in excitatory transmission (). We investigated the voltage regulation and electrogenicity of this novel transporter, using human embryonic kidney (HEK) 293 cells stably transfected with rat PROT cDNA. In physiological solutions between -140 and -40 mV, L-proline (PRO) and its six-member ring congener L-pipecolate (PIP) induced inward current. The current-voltage relationship and the variance of current fluctuations were similar for PRO- and PIP-induced current, and the ratio of induced variance to the mean current ranged from 20 to 60 fA. Des-Tyr-Leu-enkephalin (GGFL), a competitive peptide inhibitor of PROT, reduced the rat PROT-associated current to control levels. GGFL alone did not elicit currents, and the GGFL-sensitive substrate-induced current was absent in nontransfected cells. Finally, GGFL inhibited PROT-mediated transport only when applied to the extracellular face of PROT. These data suggest that (1) PROT uptake is electrogenic, (2) individual transporter currents are voltage-independent, and (3) GGFL is a nonsubstrate inhibitor that interacts either with an extracellular domain of PROT or in an externally accessible pore.

Amino Acid Transport Systems, Neutral↗
PubMed1999

Photodynamic therapy with verteporfin for choroidal neovascularization caused by age-related macular degeneration: results of a single treatment in a phase 1 and 2 study.

OBJECTIVE: To evaluate the safety and short-term visual and fluorescein angiographic effects of a single photodynamic therapy treatment with verteporfin with the use of different dosage regimens in patients with choroidal neovascularization (CNV) from age-related macular degeneration. DESIGN: Nonrandomized, multicenter, open-label, clinical trial using 5 dosage regimens. SETTING: Four ophthalmic centers in North America and Europe providing retinal care. PARTICIPANTS: Patients with subfoveal CNV caused by age-related macular degeneration. METHODS: Standardized protocol refraction, visual acuity testing, ophthalmic examination, color photographs, and fluorescein angiograms were used to evaluate the effects of a single treatment of photodynamic therapy with verteporfin. Follow-up was planned through 3 months in 97 patients and for less than 3 months in 31 other patients. RESULTS: The mean visual acuity change (and range of change) from baseline at the follow-up examination at week 12 after a single treatment with regimens 1 through 5 was -0.2 (-3 to +2), -0.9 (-9 to +5), -1.6 (-9 to +2), +0.4 (-8 to +7), and +0.1 (-8 to +9) lines, respectively. Only the highest light dose (150 J/cm2) in regimens 2 and 3, which produced angiographic nonperfusion of neurosensory retinal vessels, caused marked vision loss. Some cessation of fluorescein leakage from CNV was achieved without loss of vision when the light dose used was less than 150 J/cm2. Systemic adverse events were rare. Cessation of fluorescein leakage from CNV was noted in all regimens by 1 week after photodynamic therapy. Fluorescein leakage from at least a portion of the CNV reappeared by 4 to 12 weeks after treatment in almost all cases. Progression of classic CNV beyond the area of CNV identified before treatment was noted in 42 (51%) of the 83 eyes with classic CNV followed up for 3 months after a single treatment. Eyes in which the area of any CNV leakage at 12 weeks was less than at baseline had a significantly better visual acuity outcome (+0.8 line) than eyes in which CNV leakage progressed (-0.8 line). CONCLUSIONS: Photodynamic therapy with verteporfin achieved short-term cessation of fluorescein leakage from CNV without loss of vision or growth of classic CNV in some patients with age-related macular degeneration. Except for nonperfusion of neurosensory retinal vessels at a light dose of 150 J/cm2, no other adverse events were of concern. Randomized clinical trials to investigate whether this new modality can preserve vision in patients with CNV secondary to age-related macular degeneration are justified.

Aged↗
PubMed1999

Photodynamic therapy with verteporfin for choroidal neovascularization caused by age-related macular degeneration: results of retreatments in a phase 1 and 2 study.

OBJECTIVES: To evaluate safety and short-term visual acuity and fluorescein angiographic effects of photodynamic therapy (PDT) after retreatments with verteporfin for choroidal neovascularization (CNV) in age-related macular degeneration (AMD) that demonstrated fluorescein leakage after at least 1 course of PDT. DESIGN: Nonrandomized, multicenter, open-label phase 1 and 2 clinical trial using 2 different retreatment dosage regimens. SETTING: Four ophthalmic centers in Europe and North America providing retinal care. METHODS: Standardized protocol refraction, visual acuity testing, ophthalmic examinations, color photographs, and fluorescein angiograms were used to evaluate the results of multiple PDT treatments. Two regimens (regimens 2 and 4) for treatment and retreatment were chosen from 5 used in a single-treatment study. Both regimens used a verteporfin dose of 6 mg/m2 infused for 10 minutes. However, regimen 2 used a light dose of 100 J/cm2 applied 20 minutes after the start of the verteporfin infusion, whereas regimen 4 used a light dose of 50, 75, or 100 J/cm2 applied 15 minutes after infusion commenced. Posttreatment evaluations were planned in 31 participants up to 3 months after up to 2 retreatments given at 2- or 4-week intervals after initial PDT treatment. Similar posttreatment evaluations were planned after retreatments in 5 additional participants who were reenrolled some time more than 12 weeks after an initial PDT treatment. RESULTS: The average visual acuity change for the 31 participants who had retreatment within 2 to 4 weeks after the initial treatment and a follow-up examination 16 to 20 weeks after the initial treatment was 0.2 lines (range, -4 to 4 lines) in regimen 2 and -1.0 line (range, -5 to 3 lines) in regimen 4. Similar outcomes were noted in the 5 reenrolled participants. Cessation of fluorescein leakage from classic CNV for at least 1 to 4 weeks could be achieved without loss of visual acuity after at least 2 treatments in 2 (6.5%) of 31 patients. Similar to single-treatment effects, the disappearance of leakage was documented regularly at 1 week after each retreatment. Fluorescein leakage reappeared by 4 to 12 weeks after a retreatment in almost all cases. However, compared with baseline, leakage activity appeared to be reduced after multiple PDT courses. For the 31 patients who had follow-up for 3 months after the last retreatment and had received retreatment 2 to 4 weeks after the initial treatment, progression of CNV beyond the area identified before the retreatment was noted in 10 (48%) of the 21 eyes with classic CNV in regimen 2 and 9 (90%) of 10 eyes in regimen 4. The rate and severity of ocular or systemic adverse events were not increased by multiple applications. CONCLUSIONS: Multiple applications of PDT with verteporfin achieve repetitive, short-term cessation of fluorescein leakage from CNV secondary to AMD, without loss of visual acuity. This strategy can be used in randomized clinical trials investigating the efficacy of verteporfin in PDT for recurrent fluorescein dye leakage from persistent or recurrent CNV, following an initial or subsequent PDT treatment, with maintenance of visual acuity. Retreatments may achieve progressive cessation of leakage and prevent further growth of CNV and subsequent visual loss.

Aged↗
PubMed1999

Verteporfin photodynamic therapy retreatment of normal retina and choroid in the cynomolgus monkey.

OBJECTIVE: This study evaluated the effect of repeated photodynamic therapy (PDT) applications on normal primate retina and choroid using an intravenous infusion of liposomal benzoporphyrin derivative (verteporfin). DESIGN: This was an experimental study in a primate model. ANIMALS/CONTROLS: Six cynomolgus monkeys were used as experimental subjects and one monkey was used as a control subject. INTERVENTION: Three consecutive PDT treatments at 2-week intervals were applied over the center of the fovea or the optic nerve of each eye. Verteporfin was delivered by intravenous infusion at a dose of 6 mg/m2, 12 mg/m2, or 18 mg/m2. Laser irradiation was then applied using a diode laser (689 nm) with light doses and spot sizes kept constant. MAIN OUTCOME MEASURES: Findings were documented by fundus photography, fluorescein angiography, and light and electron microscopy. RESULTS: A cumulative dose response was seen angiographically and histologically with more severe damage to the retina and choroid noted at higher dye doses. Photodynamic therapy applied to the macula using the 6-mg/m2 verteporfin dose showed recovery of choriocapillaris, with mild retinal pigment epithelium and outer photoreceptor damage at 6 weeks. At this dose, the optic nerve showed few focal sites of axon atrophy and capillary loss. Treatments over the macula using the 12-mg/m2 and 18-mg/m2 doses led to chronic absence of choriocapillaris and photoreceptors at 6 weeks. One of two optic nerves became atrophic after PDT applications using dye doses of 12 mg/m2, and both optic nerves became atrophic in the 18-mg/m2 dye dose group. CONCLUSION: Limited damage to the retina, choroid, and optic nerve was present in primates treated with multiple PDT sessions using 6 mg/m2 verteporfin with light doses and the timing of irradiation kept constant. However, PDT using higher dye doses of 12 mg/m2 and 18 mg/m2 led to significant chronic damage to the normal retina, choroid, and optic nerve.

Animals↗
PubMed1999

Injuries and considerations in masters aquatics sports.

Masters level athletes in all aquatic sports challenge healthcare professionals, providing them with many opportunities to test their sports medicine training. Along with sports-specific injuries, the medical professional must factor in physiologic differences due to aging and even the older athlete's basic motivation to pursue the sport--which differs greatly from that of younger swimmers. Organized Masters programs, complete with national and international championships, are growing in Masters Diving, Masters Synchronized Swimming, Masters Swimming, and Masters Water Polo.

Aging↗
PubMed1999

Proton beam therapy for age-related macular degeneration: development of a standard plan.

Age-related macular degeneration is the leading cause of blindness in developing countries. Irradiating the exudative form, in which a choroidal neovascular membrane develops in the subfoveal area, is presently a treatment under investigation. In 1995, Massachusetts General Hospital, collaborating with Massachusetts Eye and Ear Infirmary, initiated a protocol to treat SCNV membranes using the proton beam at the Harvard Cyclotron Laboratory and the EYEPLAN program with a light-field setup. EYEPLAN requires the axial eye length, membrane dimensions, and manipulation of the eye to include a 4.0-mm radial margin around the membrane so that the aperture margin (50% isodose line on the posterior retina) abuts the inferior aspect of the limbus. Review of 100 individually prepared plans showed that 95% of the fabricated apertures were circular (aspect ratio < 1.095) with diameters 9.5 to 15.0 mm. This information was used to develop an automated standard plan. Thirty-nine plans were developed for axial lengths ranging from 21.0 to 25.0 mm and membrane sizes from 1.5 to 6.75 mm in the usual way as the reviewed ones. Circular targets were outlined centered on the fovea. Distal and proximal 90% ranges (modulation) to the target, and doses to macula, optic disc, lens, ciliary body, retina, and globe were calculated. An automated standard plan requiring the same input data, but avoiding the need for individual plans, was developed. The program outputs the aperture diameter, fixation angle for the light-field setup, range and modulation, and calculates dose to the macula and optic nerve and percentage of retina receiving > or = 50% and > or = 90% of the prescribed dose. Individual plans require approximately 1.5 hours; the standard plan, 5 minutes. The standard plan could have treated 86% of the reviewed plans. The automated plan provides accurate and efficient treatment parameters for the majority of patients.

Fluorescein Angiography↗
PubMed1999

Acidosis potentiates oxidative neuronal death by multiple mechanisms.

Both acidosis and oxidative stress contribute to ischemic brain injury. The present study examines interactions between acidosis and oxidative stress in murine cortical cultures. Acidosis (pH 6.2) was found to potentiate markedly neuronal death induced by H2O2 exposure. To determine if this effect was mediated by decreased antioxidant capacity at low pH, the activities of several antioxidant enzymes were measured. Acidosis was found to reduce the activities of glutathione peroxidase and glutathione S-transferase by 50-60% (p < 0.001) and the activity of glutathione reductase by 20% (p < 0.01) in lysates of the cortical cultures. Like acidosis, direct inhibition of glutathione peroxidase with mercaptosuccinate also potentiated H2O2 toxicity. Because acidosis may accelerate hydroxyl radical production by the Fenton reaction, the effect of iron chelators was also examined. Both desferrioxamine and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, two structurally different iron chelators, significantly reduced H2O2-induced neuronal death under both pH 7.2 and pH 6.2 conditions. These results suggest that the increased cell death produced by severe acidosis during cerebral ischemia may result in part from exacerbation of oxidative injury. This exacerbation may result from both impaired antioxidant enzyme functions and increased intracellular free iron levels.

Acidosis↗
PubMed1999

Homocysteine and Alzheimer's disease.

In a recent case-control study of 164 patients with clinically diagnosed Alzheimer's disease (AD), including 76 patients with the AD diagnosis confirmed postmortem, mean total serum homocysteine concentration was found to be significantly higher than that of a control group of elderly individuals with no evidence of cognitive impairment. Because homocysteine is considered an independent risk factor for vascular disease, this finding is consistent with the emerging hypothesis that vascular disease is a contributing factor in the pathogenesis of AD.

Aged↗
PubMed1999

Constitutive expression of VEGF, VEGFR-1, and VEGFR-2 in normal eyes.

PURPOSE: The expression of vascular endothelial growth factor (VEGF) and its high-affinity receptors VEGFR-1 and VEGFR-2 was investigated in normal eyes. METHODS: Monkey and rat eyes were surgically removed in animals under deep anesthesia and immediately prepared for study. Ocular VEGF, VEGFR-1, and VEGFR-2 expression was studied using a combination of in situ hybridization, northern blot analysis, immunohistochemistry, immunoassay, and reverse transcription-polymerase chain reaction. RESULTS: Steady state VEGF mRNA levels were detected in the normal vascularized ocular tissues of the monkey: the conjunctiva, iris, retina, and the choroid-retinal pigment epithelial complex. VEGF121 and VEGF165 were the major isoforms identified. VEGF protein was detected in the conjunctiva, retina, and the choroid-retinal pigment epithelial complex. Retinal VEGF mRNA localized to the ganglion, inner nuclear, and retinal pigment epithelial cell layers. VEGF protein localized to these same layers and to the cones of monkey retina. VEGFR-1 and VEGFR-2 mRNAs were detected in normal monkey iris, retina, and the choroid-retinal pigment epithelial complex. In both monkey and rat eyes, VEGFR-1 and VEGFR-2 mRNAs were localized to the inner nuclear layer of the retina. CONCLUSIONS: VEGF, VEGFR-1, and VEGFR-2 are constitutively expressed in the vascularized tissues of normal eyes.

Animals↗
PubMed1999

Effects of photodynamic therapy using verteporfin on experimental choroidal neovascularization and normal retina and choroid up to 7 weeks after treatment.

PURPOSE: To study the long-term effects of photodynamic therapy (PDT), using liposomal benzoporphyrin derivative (BPD) or Verteporfin, on experimental choroidal neovascularization (CNV) and on normal retina and choroid (with no CNV) in the cynomolgus monkey eye. METHODS: Photodynamic therapy was performed in 8 cynomolgus monkey eyes with experimental CNV induced by laser injury. The effect of PDT on normal retina and choroid (with no CNV) was studied in 9 monkey eyes. Liposomal BPD was administered intravenously (0.375 mg/kg) either as a bolus, as a slow infusion over 32 minutes, or as a fast infusion over 10 minutes. Photodynamic therapy was performed using light at a wavelength of 689 or 692 nm, with an irradiance of 600 mW/cm2 and fluence of 150 J/cm2. Follow-up studies, including fundus photography and FA, were performed at 24 hours after PDT and then weekly. Indocyanine green and BPD angiography were performed in selected cases. Tissues were examined with light and electron microscopy at the end of follow-up. RESULTS: Twenty-three of the 32 areas of CNV treated with PDT showed absence of angiographic leakage at 24 hours. Twenty-eight areas of CNV were followed for 4 weeks; 22 of 28 showed absence of angiographic leakage at 2 weeks; and 20 of 28 at 4 weeks of follow-up. Forty spots on the normal retina and choroid were treated with PDT and were followed for 4 to 7 weeks. These spots showed pigment-laden cells in the outer retina, variably pigmented retinal pigment epithelium (RPE) in the treated area, intact neurosensory retina, and reperfusion of the choriocapillaris. CONCLUSIONS: Photodynamic therapy leads to absence of angiographic leakage for at least 4 weeks in experimental CNV in the monkey model. In the normal monkey eye the RPE and choriocapillaris show generalized recovery with preservation of the neurosensory retina 7 weeks after PDT.

Animals↗
PubMed1999

Folate deficiency beyond megaloblastic anemia: hyperhomocysteinemia and other manifestations of dysfunctional folate status.

Folate plays a key role in nucleic acid synthesis. As a consequence, the most conspicuous complication of folate deficiency or of derangements of folate metabolism is megaloblastic macrocytic anemia caused by interdiction of normal proliferation of rapidly dividing bone marrow cells. Other rapidly dividing cells, including those in the gastrointestinal tract, may also be affected by the megaloblastic process. This may result in malabsorption. However, there is mounting evidence to indicate that there are other earlier manifestations of folate deficiency or of longstanding suboptimal folate nutrition. Chief among these manifestations of folate deficiency are an increased predisposition to occlusive vascular disease and thrombosis, which have been linked to increased levels of homocysteine found in folate deficiency and abnormal states of folate metabolism. In addition, folate deficiency, previously considered free of neurological consequences, is now known to be associated with disturbances of mood, and even spinal cord syndromes similar to those seen in vitamin B12 deficiency. Finally, there is both experimental and clinical evidence to suggest that folate deficiency may interfere with immunologic status and may be associated with an increased predisposition to neoplasia. Nutritional as well as genetic factors may contribute to these various nonhematological manifestations of folate insufficiency.

Anemia, Megaloblastic↗
PubMed1998

Chronic pulmonary hypertension increases fetal lung cGMP phosphodiesterase activity.

An experimental ovine fetal model for perinatal pulmonary hypertension of the neonate (PPHN) was characterized by altered pulmonary vasoreactivity and structure. Because past studies had suggested impaired nitric oxide-cGMP cascade in this experimental model, we hypothesized that elevated phosphodiesterase (PDE) activity may contribute to altered vascular reactivity and structure in experimental PPHN. Therefore, we studied the effects of the PDE inhibitors zaprinast and dipyridamole on fetal pulmonary vascular resistance and PDE5 activity, protein, mRNA, and localization in normal and pulmonary hypertensive fetal lambs. Infusion of dipyridamole and zaprinast lowered pulmonary vascular resistance by 55 and 35%, respectively, in hypertensive animals. In comparison with control animals, lung cGMP PDE activity was elevated in hypertensive fetal lambs (150%). Increased PDE5 activity was not associated with either an increased PDE5 protein or mRNA level. Immunocytochemistry demonstrated that PDE5 was localized to vascular smooth muscle. We concluded that PDE5 activity was increased in experimental PPHN, possibly by posttranslational phosphorylation. We speculated that these increases in cGMP PDE activity contributed to altered pulmonary vasoreactivity in experimental perinatal pulmonary hypertension.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Developmental changes in lung cGMP phosphodiesterase-5 activity, protein, and message.

During transitional circulation, the pulmonary vascular bed undergoes a rapid and profound reduction in both tone and vascular smooth-muscle (VSM) content. 3',5'-Guanylate cyclic monophosphate (cGMP) is a crucial mediator in the regulation of pulmonary vascular resistance (PVR) and VSM proliferation. Hydrolysis of cGMP is achieved predominately by cGMP-specific phosphodiesterases (PDEs). Among the cGMP-specific PDEs, PDE5 is quantitatively prevalent in lung tissue. We have investigated the levels of pulmonary PDE5 enzymatic activity, protein, and messenger RNA (mRNA) in ovine and mouse lung during perinatal development. We report that within 1 h following birth, PDE5 activity, protein, and mRNA levels decrease in both species, in a manner that correlates with known decreases in PVR in early transition. However, from 4 to 7 d following birth, a secondary increase in PDE5 activity, protein, and mRNA occurs in both ovine and mouse lung, suggesting a complex regulation of PVR and VSM proliferation in late perinatal development. Our data imply that PDE5 may be an important mediator in the regulation of PVR in normal and possibly in pathologic states, and may ultimately provide a basis for PDE5 inhibitors as a treatment for pulmonary hypertension.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice.

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3'-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3'-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patient and that CUG-BP/hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.

Animals↗
PubMed1997

Neuronal regulation of glutamate transporter subtype expression in astrocytes.

GLT-1, GLAST, and EAAC1 are high-affinity, Na(+)-dependent glutamate transporters identified in rat forebrain. The expression of these transporter subtypes was characterized in three preparations: undifferentiated rat cortical astrocyte cultures, astrocytes cocultured with cortical neurons, and astrocyte cultures differentiated with dibutyryl cyclic AMP (dBcAMP). The undifferentiated astrocyte monocultures expressed only the GLAST subtype. Astrocytes cocultured with neurons developed a stellate morphology and expressed both GLAST and GLT-1; neurons expressed only the EAAC1 transporter, and rare microglia in these cultures expressed GLT-1. Treatment of astrocyte cultures with dBcAMP induced expression of GLT-1 and increased expression of GLAST. These effects of dBcAMP on transporter expression were qualitatively similar to those resulting from coculture with neurons, but immunocytochemistry showed the pattern of transporter expression to be more complex in the coculture preparations. Compared with astrocytes expressing only GLAST, the dBcAMP-treated cultures expressing both GLAST and GLT-1 showed an increase in glutamate uptake Vmax, but no change in the glutamate K(m) and no increased sensitivity to inhibition by dihydrokainate. Pyrrolidine-2,4-dicarboxylic acid and threo-beta-hydroxyaspartic acid caused relatively less inhibition of transport in cultures expressing both GLAST and GLT-1, suggesting a weaker effect at GLT-1 than at GLAST. These studies show that astrocyte expression of glutamate transporter subtypes is influenced by neurons, and that dBcAMP can partially mimic this influence. Manipulation of transporter expression in astrocyte cultures may permit identification of factors regulating the expression and function of GLAST and GLT-1 in their native cell type.

ATP-Binding Cassette Transporters↗
PubMed1997

Systemic hyperoxia decreases vascular endothelial growth factor gene expression in ischemic primate retina.

OBJECTIVES: To determine whether systemic hyperoxia can reverse retinal hypoxia and decrease vascular endothelial growth factor (VEGF) gene expression in ischemic nonhuman primate retina. METHODS: Six eyes of 3 cynomolgus monkeys were studied. Retinal ischemia was induced via laser vein occlusion and confirmed with fluorescein angiography. Animals were randomly assigned to treatment with either 21% or 100% inhaled oxygen. Arterial PO2 was monitored while systemic acid-base status was maintained. An oxygen microelectrode on a micromanipulator was used to measure preretinal oxygen concentrations in ischemic and nonischemic retina in situ. RNA was isolated from fresh whole retinas, and VEGF messenger RNA levels were quantified with Northern blotting. RESULTS: The preretinal PO2 in ischemic retina was less than the PO2 in nonischemic retina in animals breathing 21% oxygen (intervascular zone PO2, 14.3+/-0.53 vs 21.8+/-0.55 mm Hg; P=.002). After 8 hours of systemic hyperoxia (arterial PO2, 512+/-18 mm Hg), the preretinal PO2 in ischemic retina increased to 166.2+/-15.6 mm Hg (21.8% oxygen) and retinal VEGF messenger RNA levels were reduced by an average of 55%. CONCLUSIONS: These data demonstrate that systemic hyperoxia can lower retinal VEGF gene expression and reoxygenate ischemic adult primate retina.

Animals↗