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Biomedical subjects

J J Moore

Publications and source records attributed to J J Moore.

At least 55 records · Page 3Linked to original sources
PubMed1988

Oxytocin activates the inositol-phospholipid-protein kinase-C system and stimulates prostaglandin production in human amnion cells.

It has been postulated that fetal hormonal signals act upon amnion to trigger labor via prostaglandin (PG) production. Human amnion epithelial cell cultures were established to test the effects of potential activators of the inositol phospholipid-protein kinase-C effector system on intracellular inositol phosphate turnover, intracellular free calcium ([ Ca2+]i), and PGE2 production. Oxytocin provoked 3-, 2.5-, and 4-fold increases in inositol triphosphate, inositol bisphosphate, and inositol monophosphate, respectively. [Ca2+]i, measured with the fluorescent dye fura-2, was stimulated by oxytocin and vasopressin (oxytocin greater than vasopressin) in a dose-dependent manner. The [Ca2+]i transient produced by oxytocin reached a peak in 15 sec, followed by a slow return to baseline over 10 min. Preincubation with phorbol 12-myristate-13 acetate (PMA) markedly blunted the oxytocin-induced transient. No [Ca2+]i transient was seen with leukotrienes, PG, serotonin, angiotensin, or alpha- or beta-adrenergic agents. PGE2 production increased 30- to 50-fold with phospholipase-C and PMA, and 10-fold with the calcium ionophore A23187. Oxytocin and vasopressin produced 10- and 3-fold PGE2 increases, respectively. Increased PGE2 production induced by PMA, oxytocin, and A23187 was first seen after 8 hr of incubation and reached maximal levels at 24 h. Minimal PGE2 stimulation occurred with agents that produced no [Ca2+]i transient. Direct activators of the inositol phospholipid-protein kinase-C system in human amnion induce large increases in PGE2 in human amnion cells. Oxytocin and vasopressin are hormonal activators of this system in these cells, as demonstrated by their effects on inositol phosphate turnover and [Ca2+]i. These hormones also increase PGE2 production and may influence labor by stimulating PGE2 production in amnion through the inositol phospholipid-protein kinase-C system.

Amnion↗
PubMed1988

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion.

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.

Amnion↗
PubMed1988

Multisystemic chromatolytic neuronal degeneration in a Cairn terrier pup.

In a four-month-old female Cairn terrier with mild episodic paraparesis, a multisystemic chromatolytic degeneration affected widespread neuronal populations in the brain and spinal cord as well as spinal, autonomic, and enteric ganglia. There was little axon degeneration or cell body loss, and the latter findings may explain the mild clinical signs. Among affected perikarya the extent and distribution of chromatolysis varied. While peripheral lysis of Nissl substance occurred often in spinal motoneurons, central chromatolysis was frequent in brain stem nuclei, and patchy Nissl loss appeared in some neurons including those in the cerebral cortex and spinal dorsal horns. Although prior studies of various chromatolytic neurodegenerations often have demonstrated characteristic massings of neurofilaments, the major, and invariable ultrastructural finding in this study was dispersion and loss of ribosomes. Neurofilamentous accumulations were observed less consistently. The clinical and pathologic findings in this pup were compared and constrasted with those made in prior studies of chromatolytic neurodegenerations.

Animals↗
PubMed1987

Myometrial desensitization after ritodrine infusion.

We have developed a model in pregnant sheep to investigate pharmacological agents used for suppression of preterm labor. This model allows repetitive determinations of uterine contractility and simultaneous measurements of myometrial receptor and postreceptor events. We used the model to study the beta-adrenergic agent ritodrine. We infused 11 pregnant sheep with ritodrine and 3 with physiological saline for 24 h. Oxytocin boluses were given before and at 5 and 22 h after onset of the infusion. Myometrial biopsies were obtained before and immediately after the infusion. After 22 h of ritodrine infusion, uterine contractility in response to the same oxytocin bolus was 50% greater than at 5 h (P less than 0.02). Myometrial membrane beta-adrenergic receptor density decreased 49% (P less than 0.005), and catecholamine-stimulated adenylate cyclase activity was reduced 70% (P less than 0.005). The model thus demonstrates that use of the beta-adrenergic agonist ritodrine in a clinically relevant manner results in tachyphylaxis of its effects on both physiological parameters and the receptor cascade system.

Adenylyl Cyclases↗
PubMed1986

Calcium-phospholipid enhanced protein phosphorylation in human placenta.

Calcium-activated, phospholipid-dependent protein phosphorylation has not been studied in placenta. Human placental cytosol was subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence of calcium and phosphatidylserine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, calcium (10(-6) M) in combination with phosphatidylserine (50 micrograms/ml) significantly enhanced (P less than 0.01) 32P incorporation into phosphoproteins having mol wt 47,000, 43,000, and 37,000. Half-maximal 32P incorporation was observed with 3.5 X 10(-7) M Ca2+ in the presence of phosphatidylserine (50 micrograms/ml). The effect of phosphatidylserine was biphasic. In the presence of Ca 10(-6) M, 32P incorporation increased to a maximum at 70 micrograms/ml of phosphatidylserine. The increase was suppressed at 150 micrograms/ml. Tetracaine caused a dose-dependent inhibition of calcium-activated, phospholipid-dependent enhancement of the three phosphorproteins. Calcium in the absence of phospholipid enhanced the phosphorylation of a protein of 98,000 mol wt. Phosphatidylserine suppressed this enhancement. Calmodulin (10(-6) M) had no detectable effect upon phosphorylation beyond that of calcium alone, but the calmodulin inhibitor R-24571 specificlly inhibited the calcium-stimulated 98,000 mol wt phosphoprotein. Calcium-activated, phospholipid-dependent phosphoproteins are present in human placental cytosol; whether calcium-activated, calmodulin-dependent phosphoproteins also are present remains a question.

Calcium↗
PubMed1985

Adenosine 3',5'-monophosphate-dependent phosphoproteins in human placenta.

cAMP modulates estrogen, hCG, and lactate syntheses by the human placenta. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins after cAMP activation of cAMP-dependent protein kinase. cAMP-dependent phosphoproteins have not been identified in the placenta. Homogenates and cytosol from term human placenta were subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence and absence of 1.0 microM cAMP. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into proteins with mol wt of 25,000, 27,000, 39,000, 45,000, 52,000, 58,000, and 73,000 (P less than 0.02). Half-maximal 32P incorporation was observed with 1.0 X 10(-7) M cAMP, which was similar to the concentration required for half-maximal histone kinase activity (8.5 +/- 2.9 X 10(-8) M). cGMP induced 32P incorporation into the same placental proteins as cAMP, but 2 orders of magnitude greater cGMP concentrations were required to achieve phosphorylation levels similar to those caused by cAMP. cAMP-dependent protein kinase inhibitor completely blocked cGMP-induced phosphorylation, even when histone protein was added. Therefore, no evidence of a cGMP-dependent protein kinase or specific cGMP-dependent phosphoproteins were detected. CaCl2 (10(-8) - 10(-4) M) had no effect on cAMP-induced 32P incorporation into the seven cAMP-dependent phosphoproteins. However calcium induced 32P incorporation into four other proteins (mol wt, 97,000, 90,000, 20,000, and 19,000). Regulation of placental metabolism by catecholamines and other hormones known to mediate intracellular cAMP or calcium levels may be accomplished by phosphorylation of these phosphoproteins.

Calcium↗
PubMed1984

Efficacy of a latex teat sealer.

In the first of two experiments, efficacy of a latex teat sealer product was tested under field conditions on 32 cows that had one side of the udder dipped with the test product and the other left as the control. Number of new coliform, staphylococcal, or streptococcal infections in treated quarters was not reduced from controls. In a second experiment, effectiveness of this product as a protective sealer was investigated on six cows dip challenged with a broth containing 1 million Escherichia coli/ml. On sealed teats the challenge was applied on top of the dried film. The challenge organism was recovered from 33% of treated quarters and 42% of control quarters 6 h after exposure. Organisms were recovered from none of the quarters by 31 h postchallenge.

Administration, Topical↗
PubMed1984

Spermine-enhanced protein phosphorylation in human placenta.

Polyamines are known to have a role in cell proliferation, differentiation, and protein synthesis. During pregnancy, major changes in polyamine levels occur in maternal serum, amniotic fluid, and placental tissue. Polyamine-activated phosphorylation has recently been proposed as a mechanism by which polyamines may regulate metabolic processes in target tissues. Polyamine-activated protein phosphorylation has not been studied in placenta. Homogenate membrane and cytosol fractions from human placenta were subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence and absence of the polyamines, spermine and spermidine, and the diamine, putrescine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, spermine (10(-3) M) significantly (P less than 0.001) stimulated 32P incorporation into phosphoproteins having molecular weights of 55,000 and 105,000. At this concentration spermidine and putrescine failed to stimulate phosphorylation. Half-maximal 32P incorporation was observed with 3.7 +/- 1.25 X 10(-4) M spermine. Polylysine enhanced the phosphorylation of phosphoproteins of the same molecular weight as those enhanced by spermine. Heparin and high Mg2+ inhibited spermine-induced phosphorylation. cAMP and Ca2+ did not stimulate phosphorylation of the spermine-dependent phosphoproteins. Spermine, however, acted as an antagonist for cAMP-dependent phosphorylation of a Mr 45,000 phosphoprotein.

Adenosine Triphosphate↗
PubMed1983

Adenosine 3',5'-monophosphate (cAMP)-binding protein and cAMP-dependent protein kinase in human placenta.

cAMP modulates estrogen, hCG, and lactate syntheses by human placenta, cAMP presumably exerts its major intracellular effect by binding to cAMP-dependent protein kinase (cAMP-PK), which, in turn, phosphorylates regulatory proteins within the target cell. cAMP binding and cAMP-PK have not been previously identified in placenta. [3H]cAMP binding to crude cytosol fractions of term placenta was rapid, saturable, and reversible. Scatchard analyses of saturation experiments of [3H]cAMP binding to placental cytosol were linear (Kd = 1.13 +/- 0.11 x 10(-8) M; n = 5). The binding capacity was 1.27 +/- 0.18 pmol/mg protein. Competition for the [3H]cAMP-binding site followed the potency order cAMP much greater than cGMP much greater than (Bu)2cAMP, analogous to cAMP binding to cAMP-PK in other tissues. ADP, ATP, and adenosine did not compete for the [3H]cAMP-binding site. cAMP significantly enhanced phosphorylation of histone protein by placental cytosol (activity ratio, 0.57 +/- 0.04; P less than 0.01). Two peaks of [3H]cAMP binding and coincident cAMP-PK activity were identified by DEAE-cellulose column chromatography of placental cytosol corresponding to classical type I and type II cAMP-PK. While the majority of the cAMP-PK was found in placental cytosol, cAMP-PK was also demonstrated in crude microsomal and microvillous brush border membranes of human placenta after solubilization with Triton X-100 (P less than 0.05). Regulation of placental function by catecholamines and other hormones known to mediate cAMP levels may be accomplished through the phosphorylation of cellular proteins by cAMP-dependent protein kinases.

Binding, Competitive↗
PubMed1982

Ontogeny of beta-adrenergic receptors in the rat lung: effects of hypothyroidism.

Beta-Adrenergic receptors were identified in membrane fractions of rat lung with the beta-adrenergic antagonists (-)-[3H]-dihydroalprenolol (-)-[3H]DHA) and (+/-)-[125I]-iodohydroxybenzylpindolol ((+/-)-[125I]HYP). Binding capacity (Bmax) for (-)-[3H]DHA increased progressively from 46 +/- 7 on day 18 of gestation to 510 +/- 70 femtomoles . mg-1 protein (mean +/- S.D.) on postnatal day 28, at which time adult Bmax was attained. An increase in (-)-[3H]DHA binding capacity of the lung was observed between postnatal days 15 and 28, during the known period of increased thyroid gland secretory activity, serum triiodothyronine (T3), and thyroxine (T4) concentrations in the rat. We therefore studied lung beta-adrenergic receptors in rat pups made hypothyroid with propylthiouracil (PTU) (in utero and postnatally) compared to normal age-matched control pups and to euthyroid pups which were treated with PTU but were also injected daily with thyroxine (T4-treated). Hypothyroid pups grew nearly normally until postnatal day 15 but grew poorly thereafter; but day 28 somatic and lung weight, lung DNA, and protein were markedly decreased in hypothyroid pups as compared to controls. Pulmonary beta-adrenergic receptors were similar in hypothyroid pups and controls on day 15, but were markedly decreased in hypothyroid pups on day 28 (294 +/- 57 versus 489 +/- 82 femtomoles . mg-1 protein in T4 treated euthyroid controls). Treatment of the hypothyroid pups with T4 on day 25 significantly increased lung beta-adrenergic receptors to near normal concentrations by day 28. We conclude that thyroid hormones or thyroid dependent factors enhance pulmonary beta-adrenergic receptor synthesis and that thyroid hormone is required for the normal postnatal maturation of the beta-adrenergic receptor system in the rat lung.

Animals↗
PubMed1982

Trophoblastic cells of the hydatidiform mole contain a beta 1-subtype adrenergic receptor.

Both beta 1- and beta 2-adrenergic receptors have been previously described in normal human placental homogenates; the cells upon whose surface membranes these receptors reside have not been identified. In order to show that a beta 1-adrenergic receptor is present on trophoblastic cells, the cells which mediate maternal-fetal transport and produce placental hormones, beta-adrenergic receptors were demonstrated in membrane fractions of human hydatidiform mole. Microscopic sections of the mole samples used demonstrated edematous villi lined by trophoblastic cells with minimal nontrophoblastic (stromal or vascular) contamination compared with placenta. (--)-[3H]Dihydroalprenolol [(--)-[3H]DHA] binding to molar membranes was reversible and saturable to a single class of sites (Kd = 0.97 +/- 0.12 nM; n = 7; maximum binding capacity, 72.9 +/- 6.4 fmol/mg protein). (--)-[3H]DHA binding was associated with catecholamine-stimulated adenylate cyclase activity. Agonist competition for the molar beta-adrenergic receptor showed the order of potency to be (--)isoproterenol much greater than norepinephrine = epinephrine, characteristic of a beta 1-adrenergic receptor subtype. Competition for (--)-[3H]DHA binding to trophoblastic membranes by the beta-adrenergic receptor subtype-specific agents metoprolol (beta 1 selective) and zinterol (beta 2 selective) was also characteristic of a homogeneous subtype of beta 1-adrenergic receptors. Because beta 1-adrenergic receptors alone were seen on trophoblast cells, the beta 2-adrenergic receptor in placenta must reside on nontrophoblastic elements (stromal or vascular endothelium). No differences in beta-adrenergic receptor binding were seen related with ploidy (2 or 3 N), the presence or absence of a fetus, or the progression of the mole to choriocarcinoma. Two choriocarcinoma cell lines, BeWo and JEG-3, however, showed no specific (--)-[3H]DHA binding. Human trophoblast contains beta 1-adrenergic receptors coupled to catecholamine-sensitive adenylate cyclase, supporting a role for catecholamines in the regulation of placental metabolism.

Adenylyl Cyclases↗
PubMed1982

Response to abomasal infusion of methionine of weaned dairy calves fed a complete pelleted starter ration based on by-product feeds.

Three abomasal infusion trials were to determine the first-limiting amino acid for nitrogen retention of postweaned Holstein bull calves fed a starter ration based on cereal by-product feeds. Ration ingredients contributed the following percentages of total amino acids: wheat middlings 38, brewer's grains 24, hominy feed 21, oat hulls 10, distiller's grains 6, and molasses 1. Dry matter of the pelleted ration contained 13 to 14% crude protein and .23 to .26% sulfur. Each trial was a replicated 4 x 4 Latin square with 9-day periods. Calves were weaned at 4 wk and infusions initiated at 5 to 6 wk of age. Abomasal infusion of the 10 essential amino acids, an isonitrogenous quantity of an equimolar mixture of alanine, asparagine, glutamate, glycine, and serine, or an isocaloric quantity of glucose indicated that essential amino acid(s) rather than nonspecific nitrogen or energy was limiting protein synthesis. Quadratic responses in urine nitrogen excretion and retained nitrogen occurred when 0, .3, .6, and 1.2 g L-methionine/kg starter ration were infused. Greatest responses occurred with .6 g methionine, which increased retained nitrogen 11 and 19% and decreased urine nitrogen 7 and 10%. The relationship between infused methionine and plasma methionine was linear. Infusions in all trials had no effect on digestibility of energy and nitrogen.

Abomasum↗
PubMed1982

Developmental aspects of alpha- and beta-adrenergic receptors.

Clearly a number of hormonal, pathologic, and maturation factors alter alpha- and beta-adrenergic receptors during development. Ontogenic changes in these receptors appear to be organ- and species-specific, occurring at distinct times during organ maturation. Developmental changes in adrenergic receptors are probably the result of a number of homologous and heterologous regulatory mechanisms. These are likely to include the effects of differentiation and aging themselves, and in fact some of the most dramatic changes in adrenergic receptor number and function occur during development. These changes are likely important to the adaptation of the developing infant and knowledge of their appearance, regulation, and function will be useful in predicting therapeutic or toxic effects of these treatments on the developing fetus and infant when exposed to adrenergic agonists or antagonists.

Animals↗
PubMed1982

Kinetic assay of beta-hydroxybutyrate in plasma with a COBAS-BIO centrifugal analyzer.

An enzymic rate method for measuring beta-hydroxybutyrate in plasma by use of beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) was adapted to the Roche COBAS-BIO centrifugal analyzer. Optimum reagent composition and reaction conditions were determined, to provide increased sensitivity and accuracy. Use of magnesium ions increased sensitivity. Comparison of results with those of a manual enzymic endpoint method involving perchloric acid protein-free filtrates of whole blood showed excellent correlation (r = 0.996). The method has good within-run and day-to-day precision. Linearity of the standard curve extends to 5000 mumol of beta-hydroxybutyrate per liter. The assay can be completed in less than 5 min and requires 15 microL of plasma and minimal reagent volumes.

3-Hydroxybutyric Acid↗
PubMed1982

Liquid-chromatographic estimation of saturated phospholipid palmitate in amniotic fluid compared with a thin-layer chromatographic method for acetone-precipitated lecithin.

The "high-performance" liquid-chromatographic method we report for measuring palmitate derived from saturated phospholipids in amniotic fluid is sensitive and precise and possesses a wide dynamic range. Palmitate from nonsurfactant sources is largely excluded. The procedure involves oxidation of the chloroform/methanol-extracted lipids with osmium tetroxide, precipitation of the resulting phosphatides with cold acetone, mild alkaline hydrolysis, derivatization of the liberated fatty acids to phenacyl esters, and reversed-phase chromatography. The method is no less convenient or more time-consuming than measurement of the lecithin/sphingomyelin ratio by thin-layer chromatography. The clinical usefulness of the procedure is indicated by results of a correlative study of 26 amniotic fluids evaluated by "lung profile" tests in a second laboratory. A value for dipalmitoyl lecithin of 13 mg/L or greater indicates fetal lung maturity.

Acetone↗