High-level resistance to ciprofloxacin in Escherichia coli.
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Anti-Infective Agents↗
Biomedical subjects
A Graham
Publications and source records attributed to A Graham.
Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway. Implications for vascular disease.
We have previously reported that plasma apolipoprotein (apo) E-containing high density lipoprotein particles have a potent anti-platelet action, apparently by occupying saturable binding sites in the cell surface. Here we show that purified apoE (10-50 microg/ml), complexed with phospholipid vesicles (dimyristoylphosphatidylcholine, DMPC), suppresses platelet aggregation induced by ADP, epinephrine, or collagen. This effect was not due to sequestration of cholesterol from platelet membranes; apoE x DMPC chemically modified with cyclohexanedione (cyclohexanedione-apoE x DMPC) did not inhibit aggregation but nevertheless removed similar amounts of cholesterol as untreated complexes, about 2% during the aggregation period. Rather we found that apoE influenced intracellular platelet signaling. Thus, apoE x DMPC markedly increased cGMP in ADP-stimulated platelets which correlated with the resulting inhibition of aggregation (r = 0.85; p < 0.01, n = 10), whereas cyclohexanedione-apoE x DMPC vesicles had no effect. One important cellular mechanism for up-regulation of cGMP is through stimulation of nitric oxide (NO) synthase, the NO generated by conversion of L-arginine to L-citrulline, binds to and activates guanylate cyclase. This signal transduction pathway was implicated by the finding that NO synthase inhibitors of distinct structural and functional types all reversed the anti-platelet action of apoE, whereas a selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (100 nM), had a similar reversing action. Direct confirmation that apoE stimulates NO synthase was obtained by use of L-[3H]arginine; platelets pretreated with apoE x DMPC produced markedly more L-[3H]citrulline (0.71 +/- 0.1 pmol/h/10(9) platelets) than controls (0.18 +/- 0.03; p < 0.05). In addition, hemoglobin which avidly binds NO also suppressed the anti-aggregatory effect, indicating that apoE stimulated sufficient production of NO by platelets for extracellular release to occur. We conclude that apoE inhibits platelet aggregation through the L-arginine:NO signal transduction pathway.
1-Methyl-3-isobutylxanthine↗
PDGF-alpha receptor and myelin basic protein mRNAs are not coexpressed by oligodendrocytes in vivo: a double in situ hybridization study in the anterior medullary velum of the neonatal rat.
Platelet-derived growth factor (PDGF) is a growth-regulatory dimer with A and B subunits. PDGF-AA, acting via PDGF receptors of the alpha-unit subtype (PDGF-alphaR), is implicated in the differentiation of oligodendrocyte precursors and in the survival of newly formed oligodendrocytes, which gradually lose expression of PDGF-alphaR. However, it is unclear whether terminally differentiated oligodendrocytes express PDGF-alphaR in vivo. To address this question, and to help clarify the role of PDGF-AA in late oligodendrocyte differentiation, we have used double in situ hybridization with digoxigenin- and fluorescein-labeled riboprobes to relate PDGF-alphaR mRNA and myelin basic protein (MBP) mRNA expression in the isolated intact anterior medullary velum (AMV) of rats ages Postnatal Day (P) 10-12 and P30-32. In parallel experiments, AMV were immunolabeled with the oligodendrocyte-specific monoclonal antibody Rip to provide information on oligodendrocyte development and the extent of myelination. At P10, the AMV contained tracts in which axons ranged from unmyelinated to fully myelinated, whereas myelination was complete in P30-32 AMV. The first oligodendrocytes to express MBP mRNA or Rip were promyelinating oligodendrocytes, which had a "star-burst" morphology and had not yet begun to form myelin sheaths. As myelination proceeded, MBP mRNA became dispersed throughout oligodendrocyte units, comprising cell somata, processes, and internodal myelin sheaths. By P30-32, MBP mRNA had been redistributed to the myelin sheaths only, reflecting a change in the site of protein synthesis in mature myelinated axon tracts. At no stage of oligodendrocyte differentiation did we observe cellular coexpression of mRNA for PDGFalphaR and MBP. Our results indicated that oligodendrocytes lost the expression of PDGFalphaR prior to gaining that of myelin gene products, and preclude an action of PDGF-AA on Rip+/MBP+ star-burst promyelinating oligodendrocytes. The spatial and temporal expression of PDGF-alphaR mRNA in the AMV was inversely related to the pattern of maturation of both myelin and oligodendrocytes, and is consistent with PDGF-alphaR being expressed by pro-oligodendrocytes. A notable finding was the high level of expression of PDGF-alphaR mRNA in the AMV of juvenile rats, localized to cell bodies within the myelinated axon tracts, strongly suggesting that oligodendrocyte precursors persisted in the mature velum.
Animals↗
Preoptic area infusions of morphine disrupt--and naloxone restores--parental-like behavior in juvenile rats.
As in the adult lactating female, opioids disrupt (and naloxone restores), parental behavior in juvenile rats (approximately 25 days of age). Because the preoptic area regulates the display of parental behavior in lactating females, we examined its parental behavior role in the juvenile rat. At 21 days of age, juvenile rats were implanted with bilateral cannulae aimed at the preoptic area using a modified Kopf stereotaxic and extrapolating from a developing-rat brain atlas [58], and divided into two groups: Initiation and maintenance. On day 25, the initiation group received bilateral infusions of either morphine (0.50 microgram), saline (0.25 microliter), or morphine plus naloxone (0.25 microgram). Thirty minutes later, they were exposed to three 1-6-day-old pups; the maintenance group was exposed to pups until they displayed 2 consecutive days of parental behavior, then infused. Morphine disrupted parental behavior in both the initiation and Maintenance groups, and naloxone restored the behavior to control/ saline levels. Parental behavior in the juvenile animal of both sexes, therefore, is under opioid regulation that parallels the adult female.
Animals↗
Illudinic acid, a novel illudane sesquiterpene antibiotic.
A novel illudane sesquiterpene antibiotic (1) with activity against methicillin-resistant Staphylococcus aureus (MRSA), has been isolated. Its MIC against MRSA was found to be 16 micrograms/mL. It also shows L-1210 cytotoxicity with an IC50 of 10-15 micrograms/mL.
Animals↗
Positional apoptosis during vertebrate CNS development in the absence of endogenous retinoids.
We have previously shown that quail embryos that develop in the absence of vitamin A have severe defects in their central nervous system. One defect is a completely missing posterior hindbrain. Here we have studied how this comes about by examining cell death using a wholemount technique. In these A- embryos we observe two narrow bands of ectopic apoptosis. One is in the mesenchyme in the region of the first somite and occurs at the 4-6 somite stage, before neural tube closure. The second band follows immediately afterwards and occurs in the neuroepithelium of the presumptive posterior hindbrain at the 6-8 somite stage. Electron microscopy shows that the dying neuroepithelial cells exhibit the characteristics of apoptosis. Rescuing the embryos by injecting retinol before gastrulation completely prevents these apoptotic events. In an effort to identify some of the genes that may be involved in the apoptotic pathway we show that Msx-2 is upregulated in the apoptotic neuroepithelium and thus may be involved, whereas Bmp-4 is not altered and thus presumably not involved. Since these apoptotic event take place at the time of specification of axial identity and segmentation in the mesenchyme and neuroepithelium we conclude that these cells die because they are wrongly specified in terms of their rostrocaudal position, a novel phenomenon which we refer to as positional apoptosis.
Age Factors↗
Cholesterol esterification is not essential for secretion of lipoprotein components by HepG2 cells.
Hepatic acyl CoA:cholesterol acyltransferase (ACAT) activity may determine storage of cholesterol and supply of cholesteryl esters for the neutral lipid core of very low density lipoprotein. Inhibition of cholesterol esterification in HepG2 cells, by the ACAT inhibitor 447C88, partially reduced the secretion of labelled total cholesterol, but the secretion of apoprotein B mass, and of radiolabelled triacylglycerol and phosphatidylcholine were unaffected. Furthermore, this compound was shown to substantially deplete the intracellular cholesteryl ester mass without affecting secretion of lipoprotein components. In contrast, the less potent ACAT inhibitor, CL277,082, significantly decreased secretion of labelled triacylglycerol, phosphatidylcholine and total cholesterol, in a manner which mirrored the decreases in secretion of apoB. This study clearly illustrates that ACAT inhibitors can exert differential effects on secretion of apoB-containing lipoproteins, which do not correlate with their efficacy in inhibiting ACAT, arguing that cholesterol esterification is not essential for lipoprotein secretion from these cells.
Anticholesteremic Agents↗
Teenagers' knowledge of emergency contraception: questionnaire survey in south east Scotland.
OBJECTIVE: To determine the level of knowledge of emergency contraception among 14 and 15 year olds. DESIGN: Confidential questionnaire survey. SETTING: 10 secondary schools in Lothian, south east Scotland. SUBJECTS: 1206 pupils predominantly (98.7%) aged 14 and 15 in the fourth year of secondary school. MAIN OUTCOME MEASURES: Knowledge of the existence of emergency contraception; of its safety, efficacy, and time limits; and of where to obtain it. RESULTS: 1121 (93.0%) fourth year pupils aged 14-16 had heard of emergency contraception. 194 girls (32.7%) and 168 boys (27.5%) had experienced sexual intercourse. Of girls who had experienced sexual intercourse, 61 (31.4%) had used emergency contraception. Knowledge of correct time limits was poor, sexually active girls being the most knowledgeable. Pupils attending schools ranked lower than the national average for academic attainment were less likely to have heard of emergency contraception and more likely to have been sexually active. 861 (76.8%) pupils knew they could obtain emergency contraception from their doctor. 925 (82.5%) pupils believed emergency contraception to be effective but 398 (35.5%) thought it more dangerous than the oral contraceptive pill. CONCLUSIONS: One third of sexually active girls aged under 16 in Lothian have used emergency contraception. This may help explain the fairly constant teenage pregnancy rates despite increasing sexual activity. Scottish teenagers are well informed about the existence of emergency contraception. However, many do not know when and how to access it properly. Health education initiatives should target teenagers from less academic schools as they are more likely to be sexually active at a young age and are less well informed about emergency contraception.
Adolescent↗
Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.
We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase.
Calcium-Calmodulin-Dependent Protein Kinases↗
Neural crest apoptosis and the establishment of craniofacial pattern: an honorable death.
During development of the vertebrate head neural crest cells emigrate from the hindbrain and populate the branchial arches, giving rise to distinct skeletal elements and muscle connective tissues in each arch. The production of neural crest from the hindbrain is discontinuous and crest cells destined for different arches, carrying different positional cues, are separated by regions of apoptosis centered on rhombomeres (r) 3 and r5. This cell death program is under the interactive control of the neighboring hindbrain segments. Both r3 and r5 produce large numbers of crest cells when freed from their flanking rhombomere, but when conjoined with their neighbor the cell death program is restored. Two key components of this program are Bmp 4 and msx-2, both of which are expressed in the apoptotic foci of r3 and r5 and which are also regulated by neighbor interactions. Importantly, the addition of recombinant Bmp 4 to isolated cultures of r3 and r5 induces the expression of Bmp 4 and msx-2 and restores the cell death program. This early neural crest segregation is maintained during development and it has profound effects upon the final craniofacial pattern. Even though crest cells from different axial origins will contribute to compound skeletal elements, these distinct populations do not intermingle. Furthermore head muscle connective tissues are exclusively anchored to skeletal domains arising from neural crest from the same axial level. Thus the discontinuous production of neural crest sculpts the crest into nonmixing streams and consequently ensures the fidelity of patterning.
Animals↗
Impaired mobilisation of cholesterol from stored cholesteryl esters in human (THP-1) macrophages.
The formation of macrophage-derived foam cells is central to the development of fatty streaks within the arterial wall, and to the progression of atherosclerosis. The unregulated deposition of cholesteryl esters, as lipid droplets within the cytoplasm of these cells, is responsible for the formation of foam cells; this process is thought to be regulated by the balance between cholesterol esterification, by acyl CoA:cholesterol acyltransferase (ACAT), and hydrolysis, by neutral cholesteryl ester hydrolase (nCEH). This study examines the importance of the balance between these two enzymes in determining the efflux of cholesterol from human (THP-1) macrophages. The presence of modified lipoprotein, or of 25-hydroxycholesterol, markedly increased cholesterol esterification in these cells and these effects were potently inhibited by the presence of the ACAT inhibitor, 447C88. In the absence of HDL, an acceptor particle, there was little or no hydrolysis of the cholesteryl ester pool and no efflux of cholesterol to the extracellular milieu; addition of HDL led to a partial (36%) reduction in cholesteryl esters, an effect which was not enhanced by the inhibition of ACAT. This suggested that the stored cholesteryl esters in human (THP-1) macrophages, unlike those in mouse peritoneal macrophages, were relatively resistant to removal by efflux to HDL. Efflux of newly synthesised free cholesterol from these macrophages was increased by HDL in a saturable manner, suggesting that the lack of reduction of stored cholesteryl esters was due to impaired mobilisation of cholesteryl esters to free cholesterol via nCEH. Indeed, nCEH activity in these macrophages was much lower than in mouse peritoneal macrophages, and appeared to be down-regulated in the presence of 25-hydroxycholesterol or modified lipoproteins; this loss of nCEH activity was prevented by the ACAT inhibitor 447C88. The efflux of stored cholesteryl esters from THP-1 macrophages therefore appears to be limited by the activity of nCEH.
Animals↗
Oxidation of human low-density lipoprotein by soybean 15-lipoxygenase in combination with copper (II) or met-myoglobin.
The enzyme 15-lipoxygenase has been implicated in the oxidation of low-density lipoprotein (LDL) in human atherosclerotic lesions. The biochemical mechanism for this oxidative process is not fully understood, and the interaction of the lipoxygenase-modified lipoprotein with metals or metalloproteins has not been explored. In the present study we have used soybean lipoxygenase to model the interaction of the enzyme with LDL and show that a direct oxygenation of fatty acids occurs, including those esterified to cholesterol, with no lag phase or change in electrophoretic mobility of the LDL particle but with some depletion of alpha-tocopherol. The enzyme-dependent oxidation may involve propagation through the release of peroxyl radicals from its active site but appears to have no requirement for free iron or copper. When lipoxygenase-treated LDL is exposed to either copper (II) or metMb, a rapid oxidation process occurs, resulting in a marked decrease in resistance to oxidation and an increase in the rate of modification to a form with increased electrophoretic mobility. This effect was not seen if lipoxygenase-treated LDL was oxidized by SIN-1, a peroxynitrite donor that oxidizes LDL with no requirement for endogenous lipid hydroperoxides. We propose that a synergistic interaction may occur between the peroxides inserted into LDL as a consequence of the enzymatic action of lipoxygenase with haem proteins or copper, which decreases the potency of the endogenous antioxidants and enhances oxidation.
Arachidonate 15-Lipoxygenase↗
Contribution of alpha-tocopherol in HDL3 to inhibition of LDL oxidation by human macrophages.
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Cell Line↗
Induction of thiol recycling by protein kinase C: a potentially pro-oxidant pathway for low-density lipoprotein oxidation.
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Alkaloids↗
Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells.
1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor.
Adenosine Triphosphate↗
Interactions between rhombomeres modulate Krox-20 and follistatin expression in the chick embryo hindbrain.
The rhombomeres of the embryonic hindbrain display compartment properties, including cell lineage restriction, genetic definition and modular anatomical phenotype. Consistent with the idea that rhombomeres are autonomous developmental units, previous studies have shown that certain aspects of rhombomere phenotype are determined early, at the time of rhombomere formation. By contrast, the apoptotic depletion of neural crest from rhombomeres 3 and 5 is due to an interaction with their neighbouring rhombomeres, involving the signalling molecule Bmp4. In this paper, we have examined whether inter-rhombomere interactions control further aspects of rhombomere phenotype. We find that the expression of Krox-20 and the repression of follistatin in r3 is dependent upon neighbour interaction, whereas these genes are expressed autonomously in r5. We further demonstrate that modulation of Krox-20 and follistatin expression is not dependent on Bmp4, indicating the existence of multiple pathways of interaction between adjacent rhombomeres. We also show that, although some phenotypic aspects of r3 are controlled by neighbour interactions, the axial identity of the segment is intrinsically determined.
Amino Acid Sequence↗
Human ocular melanocytes and retinal pigment epithelial cells differ in their melanogenic properties in vivo and in vitro.
PURPOSE: The vertebrate eye contains both melanocytes and retinal pigment epithelial (RPE) cells. Little is known of the pigmentary behaviour of these embryologically dissimilar cells. The aim of this study was to examine aspects of the pigmentary properties of both cell types in vitro and ex vivo to learn more of the function of these cells. METHODS: Sections of normal adult human eye were stained for tyrosinase related protein 1(TRP1), and cultures of RPE cells and choroidal melanocytes were examined immunocytochemically for TRP1 and 2 and enzymatically for tyrosinase activity (by assaying dopa oxidase activity). RESULTS: Over half of the choroidal melanocytes expressed TRP1 ex vivo; in contrast, a very small percentage of RPE cells were TRP1 positive. In vitro, passage 1 to 3 ocular melanocytes expressed TRP1 and TRP2 and had tyrosinase activity, which was influenced by the choice of substrate on which the cells were grown. Tyrosinase activity was highest when cells were grown on fibronectin and plastic, intermediate on laminin and lowest on vitreous extracellular matrix (ECM) containing pigment to which they attached and spread out poorly. In contrast, passage 3 RPE cells (which were unpigmented) showed little evidence of tyrosinase activity in short-term culture, irrespective of the substrate on which they were grown, and failed to express TRP1 and TRP2. When cells were grown on plastic for greater than 3 weeks in culture, a very low percentage of cells (< 0.1%) became TRP1 positive and this percentage was increased threefold if cells were cultured on laminin in the presence of bFGF. A few cells were also seen to contain pigment but cultures failed to show any tyrosinase activity. In contrast, RPE cells (but not melanocytes) showed a marked ability to take up pigment granules in vitro. CONCLUSIONS: The data suggest that normal human ocular melanocytes retain the capacity to produce pigment throughout adult life, and this can be demonstrated both ex vivo and in vitro. In contrast, we were unable to confirm that the majority of RPE cells play any significant role in active pigment production in the adult.
Adult↗
Human (THP-1) macrophages oxidize LDL by a thiol-dependent mechanism.
The oxidative modification of low-density lipoprotein by macrophages may be an important mechanism in the pathogenesis of atherosclerosis. The human monocytic leukaemic cell line THP-1, when stimulated with phorbol ester, shares many properties with human monocyte-derived macrophages. Oxidation of LDL by these cells was characterised by depletion of alpha-tocopherol, increases in thiobarbituric acid reactive substances and increases in electrophoretic mobility. The LDL particles were also converted to a form which increased accumulation of cholesteryl esters within macrophages. The oxidative mechanism appeared to be dependent upon the presence of thiols in the cellular medium. Oxidation of LDL by THP-1 macrophages, and production of thiols by these cells, were dependent upon the presence of L-cystine in the medium. Furthermore, cellular oxidation of LDL could be partially mimicked by the addition of cysteine to Hams F10 medium. Macrophage-independent oxidation of LDL, mediated by the addition of copper ions, was inhibited by cystine and cysteine in phosphate buffered saline, but not in Hams F10 medium. The glutathione content of THP-1 macrophages was also dependent upon the presence of cysteine or cystine in the medium, but inhibition of glutathione synthesis by buthionine sulfoximine did not prevent the production of thiols or the oxidation of LDL by THP-1 macrophages.
Buthionine Sulfoximine↗