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N G Anderson

Publications and source records attributed to N G Anderson.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mitogenic signalling by delta opioid receptors expressed in rat-1 fibroblasts involves activation of the p70s6k/p85s6k S6 kinase.

The regulation of mitogenic signalling pathways by G-protein-coupled receptors has been studied in Rat-1 fibroblasts stably transfected with the murine delta opioid receptor. We showed recently that stimulation of this receptor led to the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase [Burt, Carr, Mullaney, Anderson and Milligan (1996) Biochem. J. 320, 227-235]. The present study has examined the role of the ribosomal S6 kinase p70(s6k) in mitogenic signalling by the delta opioid receptor. Treatment of Rat-1 fibroblasts expressing this receptor with the synthetic enkephalin [d-Ala,d-Leu]-enkephalin (DADLE) led to a dose-dependent increase in p70(s6k) enzyme activity. Activation of p70(s6k) was dependent on the level of delta opioid receptor expressed and was sustained above basal levels for several hours. Immunoblotting revealed that p70(s6k) was subject to increased phosphorylation, the extent of which coincided temporally with enzyme activation. Activation of p70(s6k) by DADLE, but not by platelet-derived growth factor, was blocked by pretreatment of cells with pertussis toxin. Activation of p70(s6k) was also partly blocked by wortmannin, indicating that phosphoinositide 3-OH kinase is required for full activation of p70(s6k) by opioid receptor agonists. Activation of the delta opioid receptor in transfected cells led to increased DNA synthesis. This increase was prevented by rapamycin, which also completely blocked activation of p70(s6k) by DADLE. In addition, prevention of the activation of p42 and p44 MAP kinases also blocked the induction of DNA synthesis by DADLE. These results suggest that the activation of both MAP kinases and p70(s6k) might be crucial to the induction of mitogenic responses by Gi-linked receptors such as the delta opioid receptor.

Androstadienes↗
PubMed1997

Growth hormone and phorbol esters require specific protein kinase C isoforms to activate mitogen-activated protein kinases in 3T3-F442A cells.

Previous studies have shown that the activation of p44 and p42 mitogen-activated protein (MAP) kinases (ERK1 and ERK2) by growth hormone (GH) and phorbol esters, but not by epidermal growth factor, in 3T3-F442A preadipocytes is dependent on protein kinase C (PKC). In the present study two approaches have been taken to determine the PKC isoform dependence of MAP kinase activation in these cells. By immunoblotting with specific antibodies, the cells were found to express PKC-alpha, -gamma,-delta, -epsilon and -zeta. Treatment of cells with 500 nM PMA for 3 h led to the complete depletion of PKC-delta and the partial depletion of PKC-alpha but did not significantly affect the expression of the other PKC isoforms. In parallel, such treatment severely attenuated the ability of GH to activate MAP kinase. The degree of this attenuation was not increased by more prolonged PMA pretreatment, indicating that PKC-delta and perhaps PKC-alpha are important for MAP kinase activation by GH. These experiments further revealed that additional PKC isoforms were required for the full activation of MAP kinases by acute treatment with PMA. A second approach involved the use of anti-sense oligodeoxynucleotides (ODNs) to deplete the individual PKC isoforms selectively. Each of the ODNs used effectively depleted the relevant isoform to undetectable levels and did not affect the expression of the other PKC isoforms. Pretreatment of cells with PKC-delta anti-sense ODN, but not with anti-sense ODN to the other phorbol ester-sensitive isoforms, severely attenuated the activation of MAP kinases by GH. PKC-delta anti-sense ODN also blocked (by approx. 50%) the activation of MAP kinases by PMA. Furthermore a combination of PKC-delta and -epsilon anti-sense ODNs completely blocked the effect of PMA on MAP kinases. Collectively, these results indicate that the novel PKC-delta and -epsilon isoforms can couple to the MAP kinase pathway in 3T3-F442A cells but that the activation of MAP kinases by GH specifically involves PKC-delta.

3T3 Cells↗
PubMed1997

Vesicoureteric reflux in the newborn: relationship to fetal renal pelvic diameter.

There has been a low yield of primary vesicoureteric reflux (VUR) from screening the fetal urinary tract during obstetric sonography. We sought to determine whether changing the cut-off level of fetal renal pelvic diameter from 10 mm to 4 mm would improve the yield of VUR. In a prospective community-based study, a fetal renal pelvic diameter of 4 mm or more on a transverse view of the fetal renal hilum at obstetric sonography after 16 weeks' gestation was found in 426 fetuses from 9,800 consecutive pregnancies. After birth, renal sonography was performed on 386 of the 426 babies. Of the 386 babies, 264 (187 boys) had a voiding cystourethrogram (VCUG) at a mean age of 9 weeks. Primary VUR was detected in 33 (16 boys) of the 264 infants (13%), and secondary VUR in another 5 (2%). Only 5 of the 33 (15%) babies with primary VUR would have been detected if a cut-off point of 10 mm for fetal renal pelvic diameter had been used. The prevalence of reflux was similar at each cut-off level of antenatal renal pelvic diameter from 4 to 10 mm. Neither calyceal nor ureteric dilatation was helpful in differentiating those with from those without VUR. The postnatal renal sonogram did not distinguish whether reflux was present or not. More infants with primary VUR, particularly girls, were found by changing the cut-off point for fetal renal pelvic diameter from 10 mm to 4 mm, and performing a VCUG on all such infants even if the postnatal renal sonogram was normal. Of the 33 infants with primary VUR, 9 (27%, 5 boys) had an abnormal dimercaptosuccinic acid scan. Our findings support the screening of the obstetric population for a fetal renal pelvic diameter of 4 mm or more, and then investigating the infants for VUR after birth.

Female↗
PubMed1997

Agonist-mediated tyrosine phosphorylation of isoforms of the shc adapter protein by the delta opioid receptor.

Maximally effective concentrations of the opioid agonist D-ala2-D-leu5-enkephalin resulted in some 2-3-fold enhancement of tyrosine phosphorylation of the p52 Shc adapter protein in a clone of Rat-1 fibroblasts transfected to express stably the murine delta opioid receptor. More limited modifications of the tyrosine phosphorylation status of the p46 and p66 forms of Shc were observed in parallel. Epidermal growth factor caused some 10-12-fold enhancement of tyrosine phosphorylation of p52 Shc and marked increases in the p46 and p66 forms. The effect of D-ala2-D-leu5-enkephalin was prevented by pretreatment of the cells with pertussis toxin and was not observed in non-transfected parental fibroblasts whereas the effect of epidermal growth factor was still manifest in both these situations. Half-maximal effects of D-ala2-D-leu5-enkephalin on p52 Shc tyrosine phosphorylation were produced with sub-nanomolar concentrations, in agreement with previous results on the tyrosine phosphorylation of p44MAPK (Burt et al., 1996). p52 Shc became tyrosine phosphorylated more rapidly than p44MAPK in response to D-ala2-D-leu5-enkephalin and its enhanced tyrosine phosphorylation was maintained for at least 10 min.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Agonist activation of p42 and p44 mitogen-activated protein kinases following expression of the mouse delta opioid receptor in Rat-1 fibroblasts: effects of receptor expression levels and comparisons with G-protein activation.

Rat-1 fibroblasts were transfected with a cDNA encoding the mouse delta opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [D-Ala2]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5'-[gamma-[35S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42MAPK (42 kDa isoform) and p44MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but in both clones essentially full phosphorylation of both p42MAPK and p44MAPK could be achieved. EC50 values for DADLE stimulation of GTPase activity and for activation of p44MAPK were substantially lower in clone D2 than in clone DOE. Moreover, in both clones the EC50 value for DADLE stimulation of p44MAPK was substantially lower than that for stimulation of GTPase activity, and the Hill coefficients for agonist activation of p44MAPK (h > 1) displayed marked co-operativity whereas those for G-protein activation did not (h 0.8-1.0). DADLE activation of p44MAPK showed more sustained kinetics in clone D2 than in clone DOE. By contrast, lysophosphatidic acid, acting at an endogenously expressed G-protein-coupled receptor, also activated p44MAPK in both clones in a pertussis toxinsensitive manner, but both the kinetics and the concentration-response curve for activation of p44MAPK by this ligand were similar. As with other systems, maintained cellular levels of a cAMP analogue prevented the effects of both G-protein-coupled receptors on activation of p44MAPK. These results demonstrate for the first time that an opioid receptor, at least when expressed in Rat-1 fibroblasts, is able to initiate activation of the MAP kinase cascade in a G1-dependent manner, and show that only a very small proportion of the cellular G1 population is required to be activated to result in full phosphorylation of the p42MAPK and p44MAPK MAP kinases.

Animals↗
PubMed1996

Cyclic AMP stimulates the phosphorylation and activation of p42 and p44 mitogen-activated protein kinases in 3T3-F442A preadipocytes.

The effects of cyclic AMP on mitogen-activated protein (MAP) kinase activation were investigated in 3T3-F442A preadipocytes. Several agents, which raise intracellular cyclic AMP levels by distinct mechanisms, induced a transient activation of the p42 and p44 isoforms of MAP kinase and of a MAP kinase kinase. Activation of MAP kinase by cyclic AMP was prevented by two distinct inhibitors of cyclic AMP-dependent protein kinase and by PD 098059, a specific inhibitor of the activation of the MAP kinase kinase MEK 1. Therefore, in contrast to most cell types studied, cyclic AMP exerts a positive influence on the MAP kinase pathway in 3T3-F442A preadipocytes at a level upstream of the activation of MAP kinase kinase.

3T3 Cells↗
PubMed1996

Requirement for phosphoinositide 3-OH kinase in growth hormone signalling to the mitogen-activated protein kinase and p70s6k pathways.

Pituitary growth hormone (GH) co-ordinately stimulates three distinct signalling pathways in 3T3-F442A preadipocytes, the STAT (signal transducer and activator of transcription) pathway, the mitogen-activated protein (MAP) kinase cascade and p70s6k. The mechanisms linking the GH receptor to these signals have not been fully identified. In this study we have examined the role of phosphoinositide 3-OH kinase (PI 3-kinase). Pretreatment of cells with wortmannin, a specific inhibitor of PI 3-kinase, prevented the activation of p70s6k and partially inhibited the activation of p42 and p44 MAP kinases by GH. In contrast, wortmannin failed to appreciably affect the GH-stimulated tyrosyl phosphorylation of JAK-2 or STAT-1. GH transiently increased the activity of PI 3-kinase recovered in antiphosphotyrosine immunoprecipitates. In addition, several tyrosyl-phosphorylated proteins were specifically adsorbed from lysates of cells exposed to GH by a glutathione S-transferase fusion protein containing the 85 kDa regulatory subunit of PI 3-kinase. GH also induced an increase in the PI 3-kinase activity associated with both JAK-2 and insulin receptor substrate-1 (IRS-1) immunoprecipitates. These results establish PI 3-kinase as an important mediator of GH signalling to the MAP kinase and p70s6k pathways and suggest that PI 3-kinase is activated by a mechanism involving JAK-2 and IRS-1.

3T3 Cells↗
PubMed1996

Wortmannin-sensitive activation of p70s6k by endogenous and heterologously expressed Gi-coupled receptors.

In order to study the regulation of the ribosomal protein S6 kinase, p70s6k, by G protein-coupled receptors, Rat-1 fibroblasts were stably transfected with two versions of the alpha2 adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human alpha2C10 receptor, with the alpha2 agonist UK 14304 led to a transient increase in p70s6k activity. UK 14304 also activated p70s6k in a clone expressing the porcine alpha2A receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein-coupled receptors, also activated p70s6k in alpha2 receptor-transfected and in nontransfected cells. Activation of p70s6k by both UK 14304 and LPA was accompanied by increased phosphorylation of the protein. Rapamycin completely blocked the activation of p70s6k by both agents. Activation of p70s6k by UK 14304 and by LPA, but not by platelet-derived growth factor (PDGF), was blocked by preincubation of cells with pertussis toxin. Wortmannin, a selective inhibitor of phosphoinositide (PI) 3-OH kinase, prevented activation of p70s6k by UK 14304, LPA, and PDGF. These data indicate that p70s6k is regulatable by Gi-coupled receptor agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts and that activation of p70s6k by such agents appears to involve an isoform of PI 3-kinase.

Adrenergic alpha-Agonists↗
PubMed1995

Posterior fontanelle cranial ultrasound: anatomic and sonographic correlation.

OBJECTIVE: The purpose of this study was to correlate normal brain anatomy as seen on posterior fontanelle cranial sonography with anatomical sections of the premature infant brain. MATERIALS AND METHODS: Images obtained from 93 cranial ultrasound examinations performed via both the anterior and posterior fontanelle in 53 infants, ranging in gestational age from 24 to 42 weeks, were reviewed to determine the ultrasound anatomy visible and also the changing appearances with increasing gestational age. The brains of five infants were sectioned at post-mortem according to predetermined anatomical landmarks to correlate with posterior fontanelle ultrasound scan planes. Brain preservation techniques involved fixation in formalin at room temperature, refrigeration of brain following formalin fixation, and brain freezing at -17 degrees C. RESULTS: In the premature infant brain, the subarachnoid space is up to 15 mm in thickness. Occipital lobe anatomy well seen includes occipital horns of lateral ventricles, and white matter tracts to the visual cortex and visual association areas. Brain anatomy was better appreciated on sections obtained following brain freezing rather than formalin fixation. CONCLUSION: Satisfactory ultrasound anatomic correlation of the premature brain is possible using a brain freezing preservation technique. Posterior fontanelle ultrasound allows detailed illustration of occipital lobe anatomy.

Brain↗
PubMed1995

An updated two-dimensional gel database of rat liver proteins useful in gene regulation and drug effect studies.

We have improved upon the reference two-dimensional (2-D) electrophoretic map of rat liver proteins originally published in 1991 (N. L. Anderson et al., Electrophoresis 1991, 12, 907-930). A total of 53 proteins (102 spots) are now identified, many by microsequencing. In most cases, spots cut from wet, Coomassie Blue stained 2-D gels were submitted to internal tryptic digestion [2], and individual peptides, separated by high-performance liquid chromatography (HPLC), were sequenced using a Perkin-Elmer 477A sequenator. Additional spots were identified using specific antibodies.

Animals↗
PubMed1995

Pre-introductory clinical trials of Norplant implants: a comparison of seventeen countries' experience.

This report summarizes the data collected in pre-introductory Norplant implants clinical trials in 17 countries in Latin America, Asia and Africa that were coordinated by either the Population Council or Family Health International (FHI) between 1984 and 1991. A total of 16,282 women between the ages of 18 and 40 years participated in the studies with semi-annual or annual follow-up visits for up to 5 years. Gross cumulative pregnancy rates were < 0.6 per 100 women in the first year and < 1.5 in the second year in all countries. Significant differences in cumulative 5-year pregnancy rates were observed between weight groups 40-49 and 50-59 kg and between 50-59 and 60-69 kg body weight but not between 60-69 kg and those > or = 70 kg. Total cumulative discontinuation rates after five years of Norplant implants use ranged from 35.8 to 60.0 per 100 women. Younger age and low parity were associated with a higher discontinuation rate. Cumulative discontinuation rates for menstrual reasons more than doubled between the end of the first year and second year of use in 13 of 17 countries. This analysis and one previous review provide the only comparison of Norplant contraceptive system study results across a wide diversity of countries; thus allowing an appreciation of the range of clinical experience with Norplant implants and the regional differences in that experience.

Adolescent↗
PubMed1995

Diagnosis of obstructive hydronephrosis in infants: comparison sonograms performed 6 days and 6 weeks after birth.

OBJECTIVE: The purpose of this study was to compare the usefulness of renal sonograms obtained 6 days and 6 weeks after birth in differentiating obstruction from nonobstruction in patients with antenatal pyelocaliceal dilatation shown by sonography and to establish sonographic criteria to determine the degree of postnatal pyelocaliceal dilatation that warrants further investigation. MATERIALS AND METHODS: Criteria for an infant to enter the study were fetal pyelectasis of 4 mm or greater, two postnatal sonograms with the second showing persisting pyelectasis extending at least into the infundibula, and a voiding cystourethrogram showing normal findings. One hundred thirty kidneys in 100 infants met the study criteria. The first postnatal sonogram was obtained at a mean age of 6 days (range, 1-14 days) and the second at a mean age of 6.6 weeks (range, 3-16 weeks). The degree of pyelectasis was measured in the anteroposterior direction on the transverse postnatal sonograms. The diagnosis of obstruction was made by excretory urography in 99 infants and nephrostography in one infant. Kidneys were categorized as definitely obstructed, possibly obstructed (anatomic features of obstruction on excretory urogram but functionally not obstructed), or not obstructed. Receiver-operating-characteristic (ROC) curves based on renal pelvic diameters were plotted for both sonograms; the ability to detect definite obstruction or possible obstruction was compared for the two time periods; and optimal cutoff points were determined. RESULTS: The mean diameter of the renal pelvis was not significantly different between the sonogram obtained at 6 days and the sonogram obtained at 6 weeks for the 86 nonobstructed kidneys. For the 27 kidneys that were obstructed, the mean pelvic diameter increased from 18 mm (range, 5-54 mm) on the sonogram obtained at 6 days to 22 mm (range, 11-60 mm) on the sonogram obtained at 6 weeks. The mean pelvic diameter of 17 kidneys categorized as possibly obstructed increased from 6 mm (range, 0-11 mm) to 10 mm (range, 6-20 mm) between the first and second sonograms. The ROC curves for all sonograms obtained at 6 weeks provided cutoff points with greater sensitivity and specificity than did the curves for the sonograms obtained at 6 days. The optimal cutoff points were 6 mm for possible obstruction (sensitivity, 100%; specificity, 57%) and 11 mm for definite obstruction (sensitivity, 100%; specificity, 57%) and 11 mm for definite obstruction (sensitivity, 100%; specificity, 96%). CONCLUSION: Renal obstruction may be underestimated or missed on a renal sonogram obtained 6 days after birth. A sonogram obtained 6 weeks after birth is more specific for detecting obstruction.

Female↗
PubMed1994

Regulation of p42 and p44 MAP kinase isoforms in Rat-1 fibroblasts stably transfected with alpha 2C10 adrenoreceptors.

Stimulation of Rat-1 fibroblasts, stably transfected with alpha 2C10 receptors, with the specific alpha 2 agonist UK14304 led to the tyrosine phosphorylation and activation of the p42 and p44 isoforms of MAP kinase. Tyrosine phosphorylation of the MAP kinases was prevented by pertussis toxin. In unstimulated cells, there was constitutive tyrosine phosphorylation and activation of the p44 but not the p42 MAP kinase. This effect was not seen in non-transfected parental Rat-1 fibroblasts.

Animals↗
PubMed1994

Growth hormone induces the tyrosine phosphorylation and nuclear accumulation of components of the ISGF3 transcription factor complex.

Growth hormone induced the accumulation of the stat91 and p84 subunits of the transcription factor complex ISGF3 in nuclear fractions of 3T3-F442A cells. Nuclear levels of p84 and stat91 peaked 30-60 min after addition of growth hormone. Growth hormone also induced the tyrosine phosphorylation of two proteins, with similar sizes to stat91 and p84, in both nuclear and cytosolic fractions. The time course of growth hormone-induced tyrosine phosphorylation of these proteins paralleled the nuclear accumulation of stat91 and p84. Immunoprecipitation with stat91-specific antibodies confirmed that growth hormone induced the tyrosine phosphorylation of stat91 and an associated protein of M(r) = 120 kDa. These findings suggest a mechanism for the modulation of specific gene transcription by growth hormone.

3T3 Cells↗
PubMed1994

Diastematomyelia: diagnosis by prenatal sonography.

OBJECTIVE: Diastematomyelia is a form of spinal dysraphia involving a sagittal cleft in the spinal cord with splaying of the posterior spinal elements. This may be an isolated abnormality or in association with more serious dysraphic conditions. In isolated diastematomyelia, the skin is intact and the prognosis excellent. This study aimed to determine the usefulness of prenatal sonography in diagnosing diastematomyelia and in predicting the isolated form. SUBJECTS AND METHODS: The usefulness of the sonographic findings of an extra posterior echogenic focus between the fetal spinal laminae and splaying of the posterior elements for the prenatal sonographic diagnosis of diastematomyelia was assessed in five cases prospectively and in one case retrospectively. The posterior echogenic focus was first seen on prenatal sonograms at a mean age of 19 weeks' gestation (range, 18-20 weeks). The final diagnosis of diastematomyelia was made at autopsy in two cases (one with an associated meningocele), at postmortem radiography in one, at surgery in one, and at CT myelography in two. Postnatal spinal sonograms were obtained in each case. The ability to detect fetal spinal widening on prenatal sonograms was compared with the postnatal radiologic appearance on spinal plain film radiographs. RESULTS: Diastematomyelia was confirmed in all five cases where the extra posterior echogenic focus was seen at prenatal sonography. The double spinal cord was within a single dural sheath in three cases, with no bony bar present. A double dural sheath with a bony bar was noted in a fourth case. In the fifth case, a bony bar was seen on a postmortem spinal X-ray, but a double dural sheath could not be confirmed because an autopsy was not performed. In the case where diastematomyelia was not suspected on prenatal sonograms, the dural sheath was single and no bony bar was present. Splaying of the posterior elements was seen on postnatal radiographs in all cases but was suspected in only four on prenatal sonograms. Prenatal sonography accurately predicted isolated diastematomyelia in four cases and complicated diastematomyelia in one case. The one case missed prenatally was of isolated diastematomyelia. CONCLUSION: A diagnosis of diastematomyelia can be made if an extra posterior echogenic focus is seen on prenatal sonography. Splaying of the posterior elements of the fetal spine is frequently present. The pregnancy should be managed differently depending on whether the diastematomyelia is isolated, with the skin intact, or is in association with more serious neural tube defects. This distinction is possible prenatally.

Female↗