Molecular characterization of the alpha'-subunit of cone photoreceptor cGMP phosphodiesterase in normal and rd chicken.
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3',5'-Cyclic-GMP Phosphodiesterases↗
Biomedical subjects
D A Green
Publications and source records attributed to D A Green.
Molecular and biochemical analyses of iodopsin in rd chick retina.
PURPOSE: The results of previous immunocytochemical and electrophysiological studies of retinas of rd (retinal degeneration) chicks suggest that the iodopsin cone visual pigment may be defective in this mutant. The goal of this study was to determine if the primary structure and synthesis of this protein is normal in this animal model of inherited retinal degeneration. METHODS: Northern cDNA sequence and western analyses were used to study rd/rd iodopsin. cDNAs encoding rd/rd iodopsin were obtained by screening an rd/rd cDNA retinal expression library and by reverse transcription PCR. Western blots were probed with either R4 or COS-1, two different monoclonal antibodies that have been shown to specifically recognize chicken iodopsin. RESULTS: Hybridization of the +/+, +/rd, and rd/rd poly(A)+ RNA with an iodopsin cDNA probe revealed the presence of a single 1.5 kb band in each of the samples, all of which were labeled with equal intensity. No significant differences were found between the published nucleic acid sequences for normal chicken iodopsin cDNA and that determined for rd/rd iodopsin cDNA. Antibody-dependent differences in the staining intensity of the 34 kDa band containing iodopsin were observed on western blots of +/+, +/rd, and rd/rd retinal protein. R4 stained the 34 kDa band in each sample with equal intensity. COS-1 labeling of the 34 kDa band in the rd/rd sample was less intense than that observed in the +/+ and +/rd samples. CONCLUSIONS: Based on the results of the cDNA sequence and northern blot experiments, the authors conclude that the gene encoding iodopsin and transcription of this gene are normal in the rd mutant. The results of the western blot analyses of rd/rd iodopsin suggest that post-translational processing of iodopsin may be abnormal in this mutant.
Animals↗
Review of invasive cervical cancer cases for AIDS surveillance.
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Acquired Immunodeficiency Syndrome↗
Anxiolytic effects of nitrous oxide in mice in the light-dark and holeboard exploratory tests.
To investigate anxiolytic effects of nitrous oxide (N2O), male mice were tested in two exploratory models--a two-chambered light-dark (L-D) unit and a holeboard. Tests were conducted inside a glovebag through which one of three mixtures of N2O and oxygen (25, 50 and 75% N2O) or room air (RA) was circulated at a flow rate of 10 l/min. The principal findings in the L-D unit were a concentration-related increase in number of interdepartmental transitions and a generalized increase in time spent on the light side. Nitrous oxide effectively elevated transitions in the L-D unit at a lower concentration (25% N2O) than was required to increase locomotor activity in an open field (50% N2O), suggesting that these two measures are at least partially independent; transitions might reflect a specific exploratory component of locomotor behavior. In the holeboard test, a concentration-related increase in number of head dips was observed. Pretreatment with naltrexone-HCl or saline vehicle revealed a contribution by an endogenous opioid-linked locomotor stimulant effect in some measures. A dose-related reversal by flumazenil of 50% N2O-induced shifts in number of head dips and time spent head-dipping implicates a benzodiazepine receptor. Both paradigms, in particular the holeboard, should prove useful in future N2O research.
Analysis of Variance↗
Estradiol-17 beta effects on lipid metabolism of adipose tissue in nutritionally induced lean and obese ovariectomized ewes.
Nutritionally manipulated lean (68 kg) and obese (87 kg) ovariectomized ewes were administered estradiol-17 beta (E2) or sham implants. Ewes individually had ad libitum access to corn silage. Rates of de novo lipogenesis, palmitate esterification, and glycerol and fatty acid release were determined with slices of subcutaneous adipose tissue at 0, 5, and 30 d after implantation. Condition and E2 interacted over time (linear, P less than .12; quadratic, P less than .05) to affect intake. Lean ewes implanted with E2 decreased intake initially after implantation, whereas obese ewes implanted with E2 decreased intake later after implantation. The linear effect of time x condition x E2 interacted (P less than .02) for lipogenesis. Lipogenesis was inhibited in both the lean and obese ewes implanted with E2. Lean compared with obese ewes without E2 had increased lipogenesis at a faster rate over time. Esterification increased (linear, P less than .01) in the lean ewes and decreased (quadratic, P less than .01) in the obese ewes over time. A time x E2 interaction occurred for esterification (P less than .02). Glycerol and fatty acid release were variable over time within condition and E2. A 48-h adipose tissue culture determined the effect of E2 on lipid metabolism. Estradiol-17 beta decreased (P less than .05) lipogenesis, decreased (P less than .08) esterification, and maintained fatty acid and glycerol release. Data in vivo and in culture indicated that E2 acted to decrease de novo lipogenesis and palmitate esterification and had little or no effect on lipolysis.
Adipose Tissue↗
Flexion-distraction injuries to the lumbar spine associated with abdominal injuries.
Flexion-distraction injuries to the lumbar spine frequently occur with concomitant abdominal viscus injury. We encountered 16 patients with flexion-distraction lumbar spine injuries. Half of them also suffered abdominal viscus rupture. Lap safety belts were most frequently associated with these injuries; however, the incorrect, underarm use of the shoulder harness was common in our series. The diagnosis of the spinal injury was frequently delayed when abdominal viscus injury occurred together with a flexion-distraction spinal injury. Thorough physical and radiographic examination of the spine as well as a detailed history, including seat belt use, is necessary to diagnose these injuries properly.
Abdominal Injuries↗
Herniated intervertebral discs associated with unstable spinal injuries.
To examine the occurrence of traumatic herniated intervertebral discs associated with unstable spinal injuries, the authors reviewed the records of all patients with spinal cord level unstable spine injuries managed at their institution over a 26-month period. Ninety-three patients were identified. All patients had roentgenographic and computed tomographic (CT) evaluation. Magnetic resonance imaging was performed in 48 patients, and revealed the presence of a herniated intervertebral disc in 16, with the highest incidence being in the cervical spine. In the patients who had only plain film and CT scans, no disc pathology was identified. Magnetic resonance imaging provides a noninvasive means of examining intervertebral disc damage in unstable spinal injuries that might otherwise be unidentified and result in spinal cord injury at the time of surgery.
Adult↗
Insulin receptor dephosphorylation in permeabilized adipocytes is inhibitable by manganese and independent of receptor kinase activity.
Autophosphorylation of insulin receptors in digitonin-permeabilized rat adipocytes increased progressively as manganese concentrations were increased. In contrast, the percent dephosphorylation of insulin receptors, when examined by chasing the [gamma-32P]ATP with unlabeled ATP, decreased at the higher manganese concentrations. Removing manganese with EGTA increased the extent of dephosphorylation by 50 to 75%. When added only at the chase, manganese but not insulin (10(-7) M) inhibited dephosphorylation. Removal of both magnesium and manganese with EDTA completely inhibited receptor autophosphorylation. However, EDTA potentiated dephosphorylation similar to EGTA when added after 2 min of phosphorylation. Thus, a dephosphorylation reaction involving the membrane-associated insulin receptor is inhibited by manganese and is independent of the receptor kinase activity. This work suggests that manganese increases net autophosphorylation of the receptor not only by enhancing kinase activity but by inhibiting receptor dephosphorylation.
Adipose Tissue↗
Energy value of corn wet milling by-product feeds for finishing ruminants.
Five trials were conducted to evaluate the energy value of corn wet milling by-products in finishing diets. In trials 1 (45 finishing lambs, 34 kg) and 2 (70 digestion wethers, 32 kg), Rambouillet X Suffolk lambs fed corn gained faster (P less than .10), more efficiently (P less than .10) and had higher (P less than .01) digestibilities of neutral and acid detergent fiber (NDF, ADF) and starch than lambs fed dry corn gluten feed (DCGF). Lambs fed wet corn gluten feed (WCGF) consumed less feed (trial 1, P less than .05; trial 2, P less than .01), were more efficient (P less than .01) NDF and ADF digestibilities than lambs fed DCGF. Starch, NDF and ADF digestion were higher (P less than .01) for lambs fed WCGF vs wet corn bran (WCB). Lambs fed WCGF gained faster (P less than .10) and consumed more (trial 1, P = .12; trial 2, P less than .10) feed than lambs fed WCB. Dried corn bran increased (trial 1, P less than .05; trial 2, P less than .01) intake and increased (P less than .01) dry matter digestion (DMD) compared with WCB. In trial 3, rates of in vitro dry matter and NDF disappearance were similar among by-product feeds. In trial 4 (50 individually fed Shorthorn-Hereford-Angus steers, 316 kg), steers fed WCGF tended to consume more (P = .14) feed than steers fed DCGF. Corn gluten feed (CGF) replacing 0 to 46% corn decreased gain (linear, P less than .05) and DMD (linear, P less than .10), while starch digestion was highest for 23% CGF (quadratic, P less than .01). A level X CGF type interaction (P = .15) occurred for efficiency due to the lower gain of steers fed 46% DCGF. The efficiency of CGF utilization was 97% that of corn when WCGF replaced 23 or 46% corn or DCGF replaced 23% corn. Dry CGF replacing 46% corn had 87% the value of corn. In trial 5 (186 Hereford-Angus cattle, 310 kg), DCGF replacing 25 and 50% corn had 97 to 100% the efficiency of corn, while intake and gain were not affected. Dry CGF replacing 25 and 50% corn silage increased (linear, P less than .05) intake 11.3% and gain 13.8%. In ruminants, CGF is highly digestible and feed efficiency is similar to corn when WCGF is fed up to 50% of the grain component or when DCGF is fed up to 25% of the grain component.
Animal Feed↗
Direct determination of uracil in [32P,uracil-3H]poly(dA.dT) and bisulfite-treated phage PM2 DNA.
A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed. DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase. The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase. The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates. Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting. The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA. Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.
5-Methylcytosine↗
Repair of alkylated DNA: Drosophila have DNA methyltransferases but not DNA glycosylases.
DNA methyltransferase activity has been identified in crude extracts of Drosophila melanogaster pupae for the removal of methyl groups from O-6 methylguanine appearing in alkylated DNA. Additionally, N-7 methylguanine and 3 methyladenine appear to be uniquely susceptible to methyltransferase activity that resides in Drosophila pupae. Consistent with this, tests to detect DNA glycosylase activity for the repair of the latter two modified bases was unsuccessful, even though a substantial loss of methyl groups from these bases was observed. Conversely, the repair of methylated purines was not detected in extracts of Drosophila embryos. The removal of methyl groups from methylated purines was dependent upon incubation temperature and was proportional to the amount of protein added to reaction mixtures. Results indicate that the methyl group is attached to protein during the repair of methylated DNA, suggesting that it is similar to the O6-methylguanine-DNA methyltransferase identified in other organisms. Although other explanations are possible, the inability to detect DNA glycosylase activity suggests that Drosophila may not rely on base excision repair for the removal of modified or nonconventional basis in DNA.
Animals↗
The development of children's mental tracking strategies on a rotation task.
In two experiments, 5- to 13-year-old children were required to mentally track the rotation of a pointer around a circular backdrop, and to indicate the pointer's imagined position on the backdrop at the sound of a signal. In Experiment 1, children imagined the pointer rotate around its end point. In Experiment 2, children imagined the pointer rotate around its midpoint, and indicated the position of one of its two ends at the time of the signal. The end to be indicated was not specified by the experimenter until the time of the signal. Thus, children were required to use an imaging strategy that could keep track of the changing positions of both ends of the pointer. In both experiments, children older than 8 years of age generated linear distance X time functions indicating mental tracking, but younger children did not. In Experiment 2, the proportions of children at each grade level using holistic or part-to-part strategies to mentally track the pointer were comparable, as were mental tracking rates. The results were discussed in relation to both recent research on children's counting, and Piagetian and information-processing views of mental imagery development.
Child↗
Photoaffinity labeling of the beta-adrenergic receptor from cultured lymphoma cells with [125I]iodoazidobenzylpindolol: loss of the label with desensitization.
The beta-adrenergic antagonist [125I]iodoazidobenzylpindolol ( [125I]IABP) specifically photolabeled two polypeptides in membrane preparations from wild-type (WT) and coupling protein-deficient cyc- cultured lymphoma cells. The molecular weights of the two polypeptides determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis were 65,000 and 55,000. They were labeled in a ratio of approximately 1:1. Pretreatment of intact WT or cyc- cells with 1.0 microM epinephrine for 15 min (desensitization) resulted in a greater loss of the 55,000 Mr polypeptide (40-60%) relative to the 65,000 Mr peptide (10-30% loss). An 18- to 24-hr pretreatment of WT cells with terbutaline (down-regulation) led to a greater than 90% reduction of the photolabeling of both polypeptides, whereas a similar pretreatment of cyc- cells resulted in no further loss of labeled receptor than that observed after only a 15-min pretreatment with epinephrine. There was no indication of a change in the electrophoretic mobility of the [125I]IABP-labeled receptors after either short- or long-term agonist pretreatment. These data provide direct evidence for heterogeneity of the beta-adrenergic receptor in lymphoma cells. The differential loss of the [125I]IABP labeling in the two polypeptides suggests a functional heterogeneity as well.
Adenylyl Cyclases↗
Effect of dithiothreitol on the beta-adrenergic receptor of S49 wild type and cyc- lymphoma cells: decreased affinity of the ligand-receptor interaction.
Dithiothreitol (DTT) treatment of WT or cyc- lymphoma membranes resulted in the simultaneous loss of epinephrine-stimulated adenylate cyclase activity and beta-adrenergic antagonist binding. The treatment produced no significant decrease in NaF-stimulated activity and only a partial loss of the PGE1 stimulation. Epinephrine partially protected against the loss of epinephrine-stimulated cyclase activity. The decrease in beta-adrenergic stimulation of adenylate cyclase was characterized by over a 100-fold increase in Kact for epinephrine stimulation of adenylate cyclase (10 mM DTT for 30 min) with no effect on the Vmax. We have previously shown that two polypeptides (Mr = 55,000 and 65,000 daltons) are specifically labeled by [125I]iodoazidobenzylpindolol (IABP) in WT and cyc-. The IABP photolabeling of the 55,000 dalton beta-receptor polypeptide was preferentially reduced by 1.0 mM DTT, whereas 10 mM DTT eliminated the photolabeling of both polypeptides. The effects of DTT were not due to either scavenging of the nitrene or reduction of the azide. A reduction in epinephrine stimulation of adenylate cyclase was also observed after treatment of intact WT, and the effects were readily reversed in the cells by washout of the DTT. The DTT effects on membranes were not reversed by washout. Our results demonstrate that the oxidized state of the lymphoma beta-receptor is necessary for maximum sensitivity to agonist stimulation of adenylate cyclase and that low concentrations of reducing agent selectively decrease specific photo-labeling of the 55,000 dalton beta-receptor polypeptide.
Adenylyl Cyclases↗
Specific muscarinic-cholinergic desensitization in the neuroblastoma-glioma hybrid NG108-15.
The cholinergic agonist carbachol, epinephrine, and the opiate morphine all inhibit prostaglandin E1 (PGE1)-stimulated adenylate cyclase in homogenates from the neuroblastoma-glioma hybrid NG108-15. Pretreatment of the hybrid with 100 microM carbachol resulted in the rapid loss (desensitization) of the carbachol inhibition of adenylate cyclase (t1/2 less than 3 min). The desensitization of the carbachol inhibition was blocked by 0.1 microM atropine. Pretreatment with carbachol (1-24 h) did not significantly affect the inhibition of adenylate cyclase by either epinephrine or morphine, nor did it alter the PGE1-stimulated activity, that is, no supersensitization was observed. Cholate extracts of the particulate fraction from either carbachol-desensitized or of control NG108-15 were able to reconstitute adenylate cyclase activities of the coupling proteins (G/F)-deficient cyc- lymphoma cell membranes with equal efficacy. These results suggested that the coupling proteins of the adenylate cyclase were not altered by the carbachol pretreatment and that desensitization occurs at the receptor or at a receptor-associated level. However, the possibility remained that specific domains of the G/F, which interact only with muscarinic receptors, were altered.
Adenylyl Cyclases↗
beta-Adrenergic receptors and cyclic AMP responses to epinephrine in cultured human fibroblasts at various population densities.
The beta-adrenergic receptors of the intact human lung diploid fibroblast line Wl-38 and an SV-40 transformed clone of Wl-38, Wl-38-VA-13-2RA (VA13), were estimated in experiments utilizing the beta-adrenergic ligand, 125l-hydroxybenzylpindolol (125IHYP). When specific 125IHYP binding was measured in cells grown to relatively low population densities (0.15x10(6)cells/35mm dish), both Wl-38 and VA13 cells had approximately 40,000 beta-adrenergic receptors per cell. Wl-38 cells, when cultured to a high population density (0.5x10(6) cells/35/mm dish) had clearly diminished numbers of beta-adrenergic receptors and greatly decreased cAMP responses to epinephrine stimulation. On the other hand, in VA13 cells, neither the receptor number nor the beta-adrenergic response was affected by cell population density. In Wl-38 cells, the diminished cAMP response to epinephrine paralleled the decrease in number of beta-adrenergic receptors. Prostaglandin E1 (PGE1) stimulation of cAMP levels was unaffected by cell population density in either Wl-38 or VA13 cells. Thus, increased cell population density, perhaps related to density dependent inhibition of growth, caused a specific diminution in 125IHYP binding concomitant with decreased cAMP responses to epinephrine.
Cell Count↗
Differences in the forskolin activation of adenylate cyclases in wild-type and variant lymphoma cells.
The ability of the diterpene forskolin to stimulate cyclic AMP accumulation in intact cell and membrane preparations of wild-type S49 lymphoma cells (WT) and a number of variants has been confirmed. Additionally, a number of salient new findings have emerged: (a) A time delay in forskolin stimulation of cyclic AMP accumulation and adenylate cyclase (t 1/2 approximately equal to 1.5 min) occurred in all hormone-sensitive WT and variant cell and membrane preparations tested. (b) The time delay was missing in the adenylate cyclase-deficient variant (cyc-) of the S49 lymphoma cell, which also lacks functional adenylate cyclase-coupling proteins. (c) The simultaneous addition of epinephrine and forskolin to WT cells or to membrane preparations eliminated the time delay. (d) Forskolin stimulation of intact WT cells did not appear to desensitize adenylate cyclase. (e) The activation of WT adenylate cyclase by forskolin was biphasic with respect to concentration, with both high- and low-affinity components being apparent. In cyc-, only the low-affinity component was detected.
Adenylyl Cyclases↗
Direct evidence for the role of the coupling proteins in forskolin activation of adenylate cyclase.
Forskolin stimulation of adenylate cyclase in wild type (WT) S49 lymphoma membrane preparations exhibited a lag and biphasic kinetics as a function of the forskolin concentration (i.e., both a high and low affinity component were observed). In contrast to WT, forskolin stimulation of cyc- adenylate cyclase in membranes demonstrated no observable lag and only the low affinity component. Both the lag and the high affinity component characteristic of forskolin activation of WT were observed in cyc- reconstituted with cholate extracts of WT which contained the G/F protein. The potency of forskolin stimulation of reconstituted adenylate cyclase was increased still further if the reconstitution was carried out with epinephrine and Gpp(NH)p. The Vmax of the forskolin stimulation of adenylate cyclase was approximately the same in the reconstituted and unreconstituted cyc-. In addition to the experiments with reconstituted cyc-, we have demonstrated that forskolin increased the apparent affinity of epinephrine for activation of adenylate cyclase in WT, and reciprocally, epinephrine increased the apparent affinity of forskolin for activation. We conclude that the lag, biphasic kinetics of forskolin activation and the synergism of hormone and forskolin activation of WT are attributable to functional G/F and are consistent with the forskolin stabilization of the activated catalytic unit of adenylate cyclase.
Adenylyl Cyclases↗