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Biomedical subjects

N Fleming

Publications and source records attributed to N Fleming.

At least 19 recordsLinked to original sources
PubMed2000

Long-read sequence analysis of the MECP2 gene in Rett syndrome patients: correlation of disease severity with mutation type and location.

Mutations in the methyl-CpG-binding protein gene MECP2 at Xq28 cause Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder characterized by a period of stagnation followed by regression in the development of young girls. Mutations were sought in MECP2 in 48 females with classical sporadic RTT, seven families with possible familial RTT and five sporadic females with features suggestive, but not diagnostic of RTT. Long distance PCR coupled with long-read direct sequencing was employed to sequence the entire MECP2 gene coding region in all cases. Mutations were identified in 44/55 (80%) unrelated classical sporadic and familial RTT patients, but only 1/5 (20%) sporadic cases with suggestive but non-diagnostic features of RTT. Twenty-one different mutations were identified (12 missense, four nonsense and five frame-shift mutations); 14 of these were novel. All missense mutations were located either in the methyl-CpG-binding domain or in the transcription repression domain. Nine recurrent mutations were characterized in a total of 33 unrelated cases (73% of all cases with MECP2 mutations). Significantly milder disease was noted in patients carrying missense mutations as compared with those with truncating mutations ( P = 0. 0023), and milder disease was associated with late as compared with early truncating mutations ( P = 0.0190).

Amino Acid Sequence↗
PubMed2000

Maternal smoking and fetal erythropoietin levels.

OBJECTIVE: To determine the influence of maternal smoking on fetal erythropoietin concentrations in health term pregnancies and test the correlation between cotinine, a biomarker of maternal smoking, and erythropoietin levels in fetuses. METHODS: We invited women with healthy term pregnancies to participate in the study, excluding those with conditions previously known to be associated with elevated fetal erythropoietin levels. We recorded demographic data, smoking status, and labor outcome prospectively for each patient. Umbilical venous samples were collected, and serum was stored at -70C to be analyzed later for erythropoietin and cotinine. Umbilical arterial samples were tested for pH and base excess determination. We compared fetal erythropoietin and cotinine between smokers and nonsmokers and examined correlations between erythropoietin and cotinine. Kruskal-Wallis test, t test, median test, and Spearman rank correlation test were used when appropriate. Statistical significance was P <.05. RESULTS: We recruited 35 nonsmokers and 26 smokers and analyzed their samples. The two groups were comparable in demographics and birth outcomes, except for birth weights, which were lower in smokers. Fetal erythropoietin concentrations increased significantly with increasing maternal cigarette consumption, ranging from none to more than 15 cigarettes per day (P =.03). There was positive correlation between fetal erythropoietin and cotinine concentrations (r =.41; P =.04), suggesting a dose-response relationship. CONCLUSION: Fetuses of smokers had increased erythropoietin concentrations that correlate positively with fetal cotinine levels; which suggests an increased risk of subacute hypoxia related to degree of maternal cigarette consumption.

Adult↗
PubMed1999

Aluminum fluoride inhibition of cabbage phospholipase D by a phosphate-mimicking mechanism.

Aluminum fluoride (AlF(4)(-)) inhibited phospholipase D (PLD) purified from cabbage in both PIP(2)-dependent and PIP(2)-independent assays, consistent with its previously observed effect on mammalian PLD. The possibility that AlF(4)(-) may exert this effect through its known phosphate-mimicking property was examined. Inorganic phosphate, as well as two phosphate analogs, beryllium fluoride and orthovanadate, also inhibited cabbage PLD. Enzyme kinetic studies confirmed that PLD followed Hill kinetics, characteristic for allosteric enzymes, with an apparent Hill coefficient (n(app)) of 3.8, indicating positive cooperativity among multiple substrate-binding sites and suggesting possible functional oligomerization of the enzyme. AlF(4)(-) modification of PLD kinetics was consistent with a competitive mode of enzyme inhibition. It is therefore proposed that AlF(4)(-), and other phosphate analogs, inhibits plant PLD by competing with a substrate phosphate group for a substrate-binding site, thereby preventing the formation of an enzyme-phosphatidyl intermediate. This may be a conserved feature of PLD superfamily enzymes.

Aluminum Compounds↗
PubMed1999

Aluminum fluoride inhibits phospholipase D activation by a GTP-binding protein-independent mechanism.

Aluminum fluoride (AlF4-) inhibited guanine nucleotide-activated phospholipase D (PLD) in rat submandibular gland cell-free lysates in a concentration-dependent response. This effect was consistent in permeabilized cells with endogenous phospholipid PLD substrates. Inhibition was not caused by either fluoride or aluminum alone and was reversed by aluminum chelation. Inhibition of PLD by aluminum fluoride was not mediated by cAMP, phosphatases 1, 2A or 2B, or phosphatidate phosphohydrolase. AlF4- had a similar inhibitory effect on rArf-stimulated PLD, but did not block the translocation of Arf from cytosol to membranes, indicating a post-GTP-binding-protein site of action. Oleate-sensitive PLD, which is not guanine nucleotide-dependent, was also inhibited by AlF4-, supporting a G protein-independent mechanism of action. A submandibular Golgi-enriched membrane preparation had high PLD activity which was also potently inhibited by AlF4-, leading to speculation that the known fluoride inhibition of Golgi vesicle transport may be PLD-mediated. It is proposed that aluminum fluoride inhibits different forms of PLD by a mechanism that is independent of GTP-binding proteins and that acts via a membrane-associated target which may be the enzyme itself.

ADP-Ribosylation Factor 1↗
PubMed1998

Control of motility patterns in the human colonic circular muscle layer by pacemaker activity.

1. This study characterized the electrical and mechanical activities of human colonic muscle strips obtained from either the ascending, descending or sigmoid colon of patient volunteers during elective colon resections. 2. Rhythmic contractile activity was observed in colonic circular muscle strips in the absence of external stimuli. This activity persisted in the presence of atropine, phentolamine, propranolol, tetrodotoxin and Nomega-nitro-L-arginine but was abolished by nifedipine. 3. The activity of whole circular muscle (WCM) was compared with that of the myenteric half (MCM), the submucosal half (SCM) and the interior (ICM) of the circular muscle layer. WCM exhibited a prominent 2-4 contractions min-1 contractile pattern which was also present in strips of SCM. In contrast, MCM and ICM exhibited slow (0.3-0.6 contractions min-1), long duration contractions with superimposed higher frequency contractions (17-18 contractions min-1). 4. Resting membrane potential (Vm), recorded at various positions through the thickness of WCM strips did not differ and averaged -50 mV. 5. Slow waves were observed in 83 % of muscles. They averaged 12 mV in amplitude, 9.4 s in duration and had a frequency of 2-4 contractions min-1. Slow waves were greatest in amplitude near the submucosal edge and decreased with distance away from this edge. Each slow wave was associated with a transient contraction. 6. Near the myenteric edge, rapid fluctuations of Vm with a mean frequency of 18 contractions min-1 were recorded in 67 % of muscles. Spiking activity was common and was superimposed upon slow waves and rapid Vm fluctuations. 7. In summary, slow waves were identified in the human colonic circular muscle layer which arise at or near the submucosal edge. These electrical events give rise to a 2-4 contractions min-1 contractile rhythm which is characteristic of the intact muscle layer. Thus, the nature and spatial organization of pacemaker activity in the human colon bears significant resemblance to other animal models, such as the dog and pig.

Adolescent↗
PubMed1998

Activation of phospholipase D by ADP-ribosylation factor in rat submandibular acinar cells.

The hydrolysis of membrane phosphatidylcholine by the enzyme phospholipase D is a key initial step in the intracellular release of the signalling molecules phosphatidic acid, diacylglycerol and arachidonic acid. Guanine nucleotide-dependent pathway leading to PLD activation were investigated in enzymatically dispersed rat submandibular acinar cells. Guanosine 5'-O-[gamma-thio]triphosphate (GTP gamma S) caused the time- and concentration-dependent stimulation of PLD in permeabilized cells. This effect was lost in prepermeabilized cells, from which cytosolic components had been allowed to leak, but was restored when endogenous cytosol, or cytosol from platelets, was added back to such cells. PLD was also activated in cytosol-depleted cells by GTP gamma S in combination with purified ARF (ADP-ribosylation factor), a low M(r) guanine nucleotide-binding protein of the ras superfamily. Additional evidence for the involvement of ARF in PLD activation was the inhibition of carbachol- or GTP gamma S-induced stimulation of the enzyme by brefeldin A, a blocker of ARF activation; and the observed translocation of ARF from cytosol to membrane on GTP gamma S treatment in permeabilized cells. The heterotrimeric G-protein stimulator, AlFn, also activated PLD, and this response, too, was inhibited by brefeldin A, suggesting the downstream involvement of ARF in coupling AlFn action to phospholipase D elevation. PLD activation caused by both GTP gamma S and AlFn was only partially reduced after treatment of cells with U73122, a demonstrated inhibitor of phospholipase C in the Gq-coupled phosphoinositide signal-transduction pathway. It is therefore proposed that in rat submandibular mucous acinar cells, a guanine nucleotide-regulated PLD activation pathway exists that involves the sequential actions of a G heterotrimeric protein and ARF. It is further suggested that this pathway is independent of the Gq/PLC/phosphatidylinositol signal transduction system.

Animals↗
PubMed1998

Sudden infant death syndrome rates subsequent to the American Academy of Pediatrics supine sleep position.

OBJECTIVES: In April 1992, the American Academy of Pediatrics (AAP) recommended that healthy infants be positioned for sleep on their side or back to reduce the risk of Sudden Infant Death Syndrome (SIDS). The authors hypothesized three different forms of the intervention to examine the impact of the recommendation according to theory such as technology diffusion. Seasonality was included in the models to control its effect when testing. METHODS: Box and Tiao time-series intervention methodology was used to examine the effect of the AAP recommendation on SIDS rates. Sudden Infant Death Syndrome mortality data from Philadelphia and Chicago were examined separately for white and nonwhite populations over 32 quarters. RESULTS: Overall SIDS rates dropped significantly according to an abrupt effect from the intervention. However, the effect appeared to be gradually declining in Philadelphia but permanent in Chicago. In Philadelphia, a decline of 62.3% was estimated in whites in the first quarter after the intervention but decreased to only 5% in the last quarter of 1994. A decline of 35.8% was estimated in nonwhites in the first quarter after the intervention but decreased to only 9.4% in the last quarter of 1994. An abrupt and permanent decrease of 26.7% and 16.5% was found in Chicago for whites and nonwhites, respectively. CONCLUSIONS: Evidence of an abrupt adoption of the recommendation can be explained by the authority innovation decision made by the AAP. Some evidence was found that the effect is temporary, perhaps because physicians are reversing earlier decisions. The demonstrated methodology provides a powerful way to test naturally occurring interventions from quasiexperimental designs to test the impact of policy guidelines.

Chicago↗
PubMed1995

Regulation of phosphatidylinositol kinases by arachidonic acid in rat submandibular gland cells.

Phosphoinositide kinases were characterized in membrane extracts of rat submandibular gland cells. Both phosphatidylinositol (PI) 4-kinase and phosphatidylinositol-4-phosphate (PI(4)P) 5-kinase phosphorylated endogenous substrates in reactions that were linear for up to 5 min, were activated by Mg2+ and showed maximal activity around neutral pH. PI 4-kinase was stimulated by Triton X-100 at an optimal concentration of 0.22%, but the detergent had an inhibitory effect on PI(4)P 5-kinase. Arachidonic acid (AA), at concentrations greater than 100 microM, inhibited the activity of both enzymes in a dose-dependent manner. The inhibitory effect was replicated by other unsaturated fatty acids, but not by a saturated fatty acid of the sn-20 series. The nature of AA inhibition of the kinases was examined in enzyme kinetic studies with exogenous phosphoinositide and adenosine 5'-triphosphate (ATP) substrates. Lineweaver-Burk plots of PI 4-kinase activity showed that AA had no effect on the apparent Km for either PI or ATP, but that the fatty acid significantly reduced Vmax (PI) from 331 to 177 pmol.mg-1.min-1 and Vmax (ATP) from 173 to 59 pmol.mg-1.min-1. This inhibitory action was consistent for PI(4)P 5-kinase kinetics, where again, AA did not alter apparent Km values, but lowered Vmax for both PI(4)P and ATP by around 50%. Since the combination of a reduced Vmax and an unchanged Km value indicates noncompetitive enzyme inhibition, it is proposed that AA regulates phosphoinositide cycle activity in submandibular gland cells by acting as a noncompetitive inhibitor of PI 4-kinase and PI(4)P 5-kinase.

1-Phosphatidylinositol 4-Kinase↗
PubMed1995

Muscarinic regulation of phospholipase D and its role in arachidonic acid release in rat submandibular acinar cells.

The characteristics of muscarinic cholinergic-induced phospholipase D (PLD) activation, and the involvement of the enzyme in the release of arachidonic acid were examined in rat submandibular acinar cells. Carbachol produced a dose-related activation of PLD to around fivefold control values at 100 microM agonist concentration. This was associated with the appearance of free choline, phosphatidic acid and arachidonic acid, indicating that the PLD substrate was phosphatidylcholine. The response to carbachol was inhibited by 60% by U73122, a blocker of a phospholipase C (PLC) specific to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], suggesting that the cleavage of phosphatidylcholine by PLD was, at least in part, secondary to agonist-coupled hydrolysis of PtdIns(4,5)P2 by PLC. Consistent with this, PLD was also activated to levels comparable to those induced by carbachol, by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the Ca2+ mobilizer, thapsigargin, two agents that respectively mimic the activation of protein kinase C (PKC) by diacylglycerol and the elevation of cytosolic Ca2+ by inositol 1,4,5-triphosphate [Ins(1,4,5)P3] in the phosphoinositide effect. The cell-permeant Ca2+ chelator 1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) abolished the thapsigargin-induced activation of PLD and inhibited the responses of PLD to carbachol and TPA by 60%. The PKC inhibitor, Ro-31-8220, also inhibited the activation of PLD by carbacol and TPA to a level of approximately double control values, but had no effect on the thapsigargin-induced elevation of PLD. A role for both the PKC-associated and Ca(2+)-mobilizing arms of the PtdIns(4,5)P2-PLC pathway in PLD regulation is thus suggested. Pretreatment of cells with the phosphatidate phosphohydrolase blocker, propranolol, significantly enhanced the carbachol-induced elevation of phosphatidic acid, but decreased agonist-stimulated production of diacylglycerol and arachidonic acid, indicating that phosphatidlycholine was the likely source of arachidonic acid. We therefore propose that, in submandibular mucous acinar cells, muscarinic activation of the PtdIns(4,5)P2-PLC pathway regulates phosphatidylcholine-specific PLD through both the PKC- and Ca(2+)-mobilizing arms of the phosphoinositide response, and that diacylglycerol, derived from phosphatidylcholine via phosphatidic acid, is a source of free arachidonic acid.

Animals↗
PubMed1995

Distribution and translocation of isoforms of protein kinase C in rat submandibular acinar cells.

The distribution of six isoforms of protein kinase C (PKC) in seromucous acinar cells of rat submandibular gland was examined and their translocation from the cytosolic- to the membrane fraction after different stimuli investigated. Western blotting, immunostaining with isoform-specific antibodies and scanning densitometry showed that PKC-alpha and epsilon were distributed fairly evenly between the cytosol and membranes in resting cells, while isoforms- beta, delta and zeta were all predominantly localized (over 80%) in membranes. PKC-gamma was not detected. PKC-alpha was mobilized to the membrane fraction by the phorbol ester, TPA, but not by the phosphoinositide-coupled agonists carbachol, methoxamine and substance P (SP). PKC-epsilon was translocated by TPA and carbachol but not by SP or methoxamine. Biochemical assay of total PKC confirmed that cytosolic enzyme activity was significantly reduced by TPA and carbachol to 29% and 75% respectively of control levels. These results suggest that muscarinic regulation of the mucosecretory response in the rat submandibular gland may be mediated by the PKC-epsilon isoform.

Animals↗
PubMed1995

Arachidonic acid stimulates intracellular calcium mobilization and regulates protein synthesis, ATP levels, and mucin secretion in submandibular gland cells.

Earlier observations that arachidonic acid inhibited the synthesis of membrane inositol phospholipids in rat submandibular acinar cells prompted the present study on whether the fatty acid may also regulate other key physiological processes in the model. Arachidonate, at concentrations above 10 mumol/L, inhibited up to 97% protein synthesis in acinar cells. The acid also lowered cellular ATP levels to 25% of control values by a ouabain-insensitive mechanism. In endoplasmic reticulum-calcium studies in permeabilized cells, arachidonic acid stimulated the mobilization of up to 73% loaded ER-45Ca2+ to the cytosol, a much greater response than those caused by other calcium translocators, thapsigargin or inositol 1,4,5-trisphosphate. Additionally, arachidonate provoked the release of over 80% of total cell 45Ca2+ to the extracellular space in intact cells and stimulated mucin secretion in the submandibular model. The inhibitory effect of arachidonic acid on protein synthesis was duplicated by carbachol, thapsigargin, and BAPTA/AM, three agents that cause net efflux of ER-Ca2+ by different mechanisms. Furthermore, comparable with the arachidonate effect on ATP, carbachol and thapsigargin also significantly reduced cellular levels of the nucleotide. It is concluded that arachidonic acid acts as a regulator of central synthetic/secretory processes in mucous acinar cells of rat submandibular gland and suggested that at least some of its effects may be secondary to its calcium-mobilizing action.

Adenosine Triphosphate↗
PubMed1992

Regulation of the cAMP signal transduction pathway by protein kinase C in rat submandibular cells.

Treatment of rat submandibular acinar cell extracts with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the dose-dependent activation of protein kinase C (PKC), assessed by the phosphorylation of a novel and highly specific substrate. This effect was duplicated by a diacylglycerol, but not by the 4 alpha-phorbol ester 4 alpha-phorbol 12,13-didecanoate. The TPA elevation of PKC was blocked by the PKC inhibitors H-7 and sangivamycin. In intact cells, TPA caused the translocation of PKC from cytosol to membrane, consistent with its known mode of activation. The beta-adrenergic agonist, isoproterenol, stimulated cAMP levels which were significantly reduced by preactivation of PKC. This inhibitory PKC effect was reversed by H-7. When cAMP was stimulated at the post-receptor level, however, by forskolin, NaF or GTP[gamma S], PKC did not inhibit, but rather enhanced the cyclic nucleotide response. Since PKC phosphorylated an endogenous protein of 55 kDa, the size of the beta 1 receptor, these findings indicate that, as in other cell types, PKC can desensitize adenylate cyclase by direct phosphorylation of the beta receptor, but potentiate the cAMP response by a post-receptor mechanism. In mucin secretion studies in the model, TPA alone caused the cAMP-independent release of up to 44% total mucin, which was much less than additive with the isoproterenol response. When the cAMP-mucosecretory response was stimulated at the adenylate cyclase level by forskolin, however, the TPA + forskolin effects were additive. These findings on the modulation of cAMP by PKC indicate cross-talk regulation in the phosphoinositide-cAMP signal transduction pathways in submandibular acinar cells.

Animals↗
PubMed1992

Arachidonic acid regulates the phosphoinositide signal transduction pathway in submandibular acinar cells.

Modulation of the phosphoinositide signal transduction pathway by arachidonic acid (AA) in collagenase-dispersed rat submandibular acinar cells was investigated. The muscarinic agonist, carbachol, stimulated PIP2 hydrolysis and the generation of IP3 to five-fold the control levels. This response was inhibited by 75% on pre-treatment of cells with AA. The AA inhibitory effect was not duplicated by a range of prostaglandins and leukotrienes and was not reversed by blockers of the cyclo-oxygenase and lipoxygenase synthetic pathways, indicating that AA action was not mediated by eicosanoid metabolites. Additional experiments confirmed that the enzyme, protein kinase C, was also not a mediator of the AA effect. Arachidonic acid did not affect the uptake of radioactive inositol into acinar cells, but it did inhibit the incorporation of inositol into inositol phospholipids of the phosphoinositide cycle. In studies on inositol phospholipid turnover, AA alone reduced the level of PIP2 but not of PIP or PI. Under conditions of PI cycle stimulation with carbachol, AA significantly lowered PIP2 and PIP but not PI. These findings suggest that arachidonic acid may regulate the phosphoinositide response by inhibiting the synthetic phase of the cycle at a locus distal to PI generation.

Animals↗
PubMed1990

Muscarinic M3 receptors are coupled to two signal transduction pathways in rat submandibular cells.

The receptor subtypes involved in muscarinic-induced phosphoinositide hydrolysis and adenylate cyclase inhibition in rat submandibular acinar cells were characterized by comparing the inhibitory potencies of four muscarinic antagonists on the two signal transduction responses. Carbachol-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis was inhibited by all antagonists with a potency rank order of 4-diphenylacetoxy-N-methyl piperidine methobromide (4-DAMP) = atropine much greater than pirenzepine much greater than AF-DX 116 (P less than 0.01). The same rank order was observed in antagonist-reversal of the reduction of cAMP caused by carbachol in the model. These findings suggest that muscarinic effects are mediated by M3 receptors in both the phosphoinositide and adenylate cyclase pathways in the submandibular gland.

Animals↗
PubMed1989

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland.

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.

Adenylate Cyclase Toxin↗
PubMed1989

Training induced physiological and metabolic changes associated with improvements in running performance.

The purpose of the present study was to examine the relationship between improvements in running performance and some of the prominent physiological and metabolic adaptations to endurance exercise training. Twelve male undergraduates agreed to participate in this study (trial group), aged matched physical education students provided a control group. Running performance, assessed as a five km time trial, improved from 19.69 +/- 2.24 to 19.22 +/- 2.03 min in the trial group (P less than 0.01) after training. Maximal oxygen uptake values increased from 56.0 +/- 6.1 to 60.7 +/- 5.4 ml.kg-1.min-1, the running speed equivalent to a blood lactate reference concentration of 4 mmol.l-1 (V-4 mM) increased from 3.79 +/- 0.77 to 4.04 +/- 0.71 m.s-1, and the rate of oxygen consumption at 3.58 m.s-1 (running economy) increased from 43.3 +/- 3.2 to 45.0 +/- 3.4 ml.kg-1.min-1 (P less than 0.01). The control group did not show any significant changes. The improved five km times in the trial group were significantly correlated (r = -0.71; P less than 0.01) with changes in the running economy rather than changes in the VO2 max (r = -0.07; ns), or V-4 mM (r = -0.13; ns) suggesting the increased rate of oxygen utilization reflected a greater oxidative degradation of metabolic substrates together with a slower rate of lactate production.

Humans↗