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M Lobban

Publications and source records attributed to M Lobban.

14 recordsLinked to original sources
PubMed1996

Rapid regulation of PDE-2 and PDE-4 cyclic AMP phosphodiesterase activity following ligation of the T cell antigen receptor on thymocytes: analysis using the selective inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and rolipram.

The PDE2, cyclic GMP-stimulated, and the PDE4, cyclic AMP-specific enzymes provide the major, detectable cyclic AMP phosphodiesterase activities in murine thymocytes. In the absence of the cyclic GMP, PDE4 activity predominated (approximately 80% total) but in the presence of low (10 microM) cyclic GMP concentrations, PDE2 activity constituted the major PDE activity in thymocytes (approximately 80% total). The PDE4 selective inhibitor rolipram dose-dependently inhibited thymocyte PDE4 activity (IC50 approximately 65 nM). PDE2 was dose-dependently activated (EC50 approximately 1 microM) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) (IC50 approximately 4 microM). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies using separated PDE1, PDE2, PDE3 and PDE4 species from hepatocytes as well as human PDE2 and PDE4 enzymes. EHNA completely ablated the ability of cyclic GMP to activate PDE2 activity, whilst having a much smaller inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operative effect were seen in the absence of cyclic GMP with Hill coefficients of approximately 0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) there was a transient decrease (approximately 83%) in PDE-4 activity and in PDE2 activity (approximately 40%). Both anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was followed by a sustained and profound increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that, dependent upon the intracellular concentrations of cyclic GMP, thymocyte cyclic AMP metabolism can be expected to switch from being under the predominant control of PDE4 activity to that determined predominantly by PDE2 activity. These activities may be rapidly and differentially regulated following ligation of different cell surface receptors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family.

COS-7 cells were transfected with a plasmid encoding a putative splice variant of PDE4A cyclic AMP-specific phosphodiesterase, RPDE-6 (RNPDE4A5). This led to the expression of a novel, cyclic AMP-specific, rolipram-inhibited phosphodiesterase activity. In such transfected cells a novel approximately 109 kDa species was recognized by anti-peptide sera raised against a dodecapeptide whose sequence is found at the extreme C-terminus of both RPDE-6 and another PDE4A splice variant. RD1 (RNPDE4A1A). RPDE-6 activity and immunoreactivity was found distributed between both pellet (approximately 25%) and cytosol (approximately 75%) fractions of transfected COS-7 cells. Soluble and pellet RPDE-6 activities exhibited similar low Km values for cyclic AMP (approximately 2.4 microM) and were both inhibited by low concentrations of rolipram, with IC50 values for the soluble activity being lower (approximately 0.16 microM) than for the pellet activity (approximately 1.2 microM). Pellet RPDE-6 was resistant to release by either high NaCl concentrations or the detergent Triton X-100. Probing brain homogenates with the anti-(C-terminal peptide) sera identified two immunoreactive species, namely an approximately 79 kDa species reflecting RD1 and an approximately 109 kDa species that co-migrated with the immunoreactive species seen in COS cells transfected to express RPDE-6. The approximately 109 kDa species was found distributed between both the low-speed (P1) and high-speed (P2) pellet fractions as well as the cytosol fractions derived from both brain and RPDE-6-transfected COS cells. In contrast, RD1 was found exclusively in the P2 fraction. Phosphodiesterase (PDE) activity immuno-precipitated by these antisera from brain cytosol had the characteristics of COS cell-expressed RPDE-6 with KmcyclicAMP approximately 3.7 microM and IC50rolipram approximately 0.12 microM. The distribution of PDE activity immunoprecipitated from the cytosol of various brain regions paralleled that seen for the distribution of the approximately 109 kDa immunoreactive species. It is suggested that the 109 kDa species identified in brain cytosol and pellet fractions is the native form of RPDE-6. The PDE4A splice variants, RD1 and RPDE-6, were shown to have distinct patterns of expression among various brain regions. PDE4A and PDE4B activities appear to provide the major source of PDE4 activity in brain membranes, whereas the cytosolic PDE4 activity is suggested to reflect predominantly the activity of the PDE4D family. Alternative splicing of the PDE4A gene confers distinct N-terminal domains on RPDE-6 and RD1, which attenuates the Vmax. of these enzymes and defines their distinct subcellular distribution pattern.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum.

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Increasing incidence of anorexia nervosa in the female population of northeast Scotland.

OBJECTIVE: The aim of the study was to determine whether there has been an increase in the incidence of anorexia nervosa in the female population in the northeast of Scotland since the 1960s. METHOD: Standardized diagnostic criteria were applied to the case records of female subjects who had been diagnosed as suffering from anorexia nervosa and had presented for the first time to psychiatric services between 1965 and 1991 or had been admitted for the first time to a general hospital between 1970 and 1991. Age-standardized annual incidence rates were calculated from 1965 through 1991. As a broad measure of severity, the weights of patients in 3 early years of the study, 1970-1972, were compared with those of patients in the last 3 years, 1989-1991. RESULTS: Over the 27-year period studied, 287 patients received confirmed diagnoses of anorexia nervosa, and the mean annual increase in incidence was 5.3%. The rate of increase was highly statistically significant. Comparison of weights at presentation showed a trend for patients presenting in 1970-1972 to be lighter than those presenting in 1989-1991. CONCLUSIONS: Rates of referral for female subjects with anorexia nervosa have greatly increased since the 1960s. These rates likely reflect a genuine increase in incidence, but the data suggest that less severely ill patients are now being referred.

Adolescent↗
PubMed1994

Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions.

In order to detect the two splice variant forms of type-IVB cyclic AMP phosphodiesterase (PDE) activity, DPD (type-IVB1) and PDE-4 (type-IVB2), anti-peptide antisera were generated. One set ('DPD/PDE-4-common'), generated against a peptide sequence found at the common C-terminus of these two PDEs, detected both PDEs. A second set was PDE-4 specific, being directed against a peptide sequence found within the unique N-terminal region of PDE-4. In brain, DPD was found exclusively in the cytosol and PDE-4 exclusively associated with membranes. Both brain DPD and PDE-4 activities, isolated by immunoprecipitation, were cyclic AMP-specific (KmcyclicAMP: approximately 5 microM for DPD; approximately 4 microM for PDE-4) and were inhibited by low rolipram concentrations (K1rolipram approximately 1 microM for both). Transient expression of DPD in COS-1 cells allowed identification of an approx. 64 kDa species which co-migrated on SDS/PAGE with the immunoreactive species identified in both brain cytosol and membrane fractions using the DPD/PDE-4-common antisera. The subunit size observed for PDE-4 (approx. 64 kDa) in brain membranes was similar to that predicted from the cDNA sequence, but that observed for DPD was approx. 4 kDa greater. Type-IV, rolipram-inhibited PDE activity was found in all brain regions except the pituitary, where it formed between 30 and 70% of the PDE activity in membrane and cytosolic fractions when assayed with 1 microM cyclic AMP, PDE-4 formed 40-50% of the membrane type-IV activity in all brain regions save the midbrain (approx. 20%). DPD distribution was highly restricted to certain regions, providing approx. 35% of the type-IV cytosolic activity in hippocampus and 13-21% in cortex, hypothalamus and striatum with no presence in brain stem, cerebellum, midbrain and pituitary. The combined type-IVB PDE activities of DPD and PDE-4 contributed approx. 10% of the total PDE activity in most brain regions except for the pituitary (zero) and the mid-brain (approx. 3%. The isolated cDNAs for DPD and PDE-4 appear to reflect transcription products which are expressed in vivo in brain. The unique N-terminal domain of PDE-4 is suggested to target this PDE to membranes in brain. Type-IVB PDEs are differentially expressed in various brain regions, indicating that there are tissue-specific controls on both the expression of the gene and the splicing of its products.

3',5'-Cyclic-AMP Phosphodiesterases↗