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Biomedical subjects

A Graham

Publications and source records attributed to A Graham.

At least 109 records · Page 6Linked to original sources
PubMed1997

Characterisation of ACTH peptides in human skin and their activation of the melanocortin-1 receptor.

Alpha-Melanocyte-stimulating hormone (alpha-MSH) is a proopiomelanocortin (POMC)-derived peptide, which is produced in the pituitary and at other sites including the skin. It has numerous effects and in the skin has a pigmentary action through the activation of the melanocortin-1 (MC-1) receptor, which is expressed by melanocytes. Recent evidence suggests that the related POMC peptides such as adrenocorticotrophin (ACTH), which is the precursor of alpha-MSH, is also an agonist at the MC-1 receptor. By using immunocytochemistry, we confirmed the presence of alpha-MSH in human skin where staining was evident in keratinocytes and especially strong in melanocytes and possibly Langerhans cells. ACTH was also present and tended to show the strongest reaction in differentiated keratinocytes. Immunostaining was also observed for the prohormone convertases, PC1 and PC2, which are involved in the formation of ACTH and its cleavage to alpha-MSH, respectively. The amounts of immunoreactive ACTH exceeded those of alpha-MSH. Using HPLC we identified for the first time the presence of ACTH1-39, ACTH1-17, ACTH1-10, acetylated ACTH1-10, alpha-MSH, and desacetyl alpha-MSH in epidermis and in cultured keratinocytes. The ability of these peptides to activate the human MC-1 receptor was examined in HEK 293 cells that had been transfected with the receptor. All peptides increased adenylate cyclase in these cells with the following order of potency: ACTH1-17 > alpha-MSH > ACTH1-39 > desacetyl alpha-MSH > acetylated ACTH1-10 > ACTH1-10. ACTH1-17 also increased the dendricity and melanin content of cultured human melanocytes indicating that the peptide was able to activate MC-1 receptors when present in their normal location. However, as found with alpha-MSH, not all cultures were responsive and, as we have previously suggested, we suspect that this was the result of changes at the MC-1 receptor. Nevertheless, it would appear that ACTH peptides can serve as natural ligands of the MC-1 receptor on human melanocytes and their presence in the skin suggests that, together with alpha-MSH, they may have a role in the regulation of human melanocytes.

Adrenocorticotropic Hormone↗
PubMed1997

Agouti protein inhibits the production of eumelanin and phaeomelanin in the presence and absence of alpha-melanocyte stimulating hormone.

Melanocytes synthesise two types of melanin: the brown-black eumelanin and the red-yellow phaeomelanin. In mice, the relative proportions of these two melanins are regulated by alpha-MSH, which preferentially increases the synthesis of eumelanin and by the Agouti protein (AP), the expression of which correlates with the growth of yellow phaeomelanin-containing hair. It has been proposed that AP acts by antagonizing the action of alpha-MSH at the MC1 receptor, although it has been suggested that it may also act independently of alpha-MSH. In the present study we show that AP inhibits melanogenesis in B16F1 melanoma cells in the presence and absence of alpha-MSH and also causes dose-related decreases in the synthesis of both eumelanin and phaeomelanin. In the presence of alpha-MSH AP had a greater effect on eumelanin production and this is consistent with an antagonistic action at the MC1 receptor. In the absence of alpha-MSH however, AP produced similar reductions in the synthesis of both melanins. These changes were not seen in B16G4F cells which lack the MC1 receptor, suggesting that even in the absence of alpha-MSH AP acts at the MC1 receptor. How this action is mediated at the intracellular level is not yet clear, although it appears to be associated with a decrease in tyrosinase activity.

Adenylyl Cyclases↗
PubMed1997

Resistance to ciprofloxacin in pathogenic Enterobacteriaceae in England and Wales in 1996.

In 1996, 6% of Escherichia coli from extraintestinal infections were resistant to ciprofloxacin with minimum inhibitory concentrations (MICs) > or = 2 mg/l (high level resistance). Low level resistance (MIC 0.125-1 mg/l) was also identified in 7% of Salmonella typhi, 4% of S paratyphi A, and 4% of non-typhoidal salmonellas. However, resistance to ciprofloxacin was rarely identified in shigellas. For E coli, physicians should be aware that treatment failures may occur when patients with invasive illness are treated with ciprofloxacin before the results of laboratory sensitivity tests are available. For salmonellas an increasing number of treatment failures have been recorded for patients infected with strains with low level resistance. Because of the increasing incidence of Enterobacteriaceae with low level resistance to ciprofloxacin, it is recommended that for this group of organisms a breakpoint of 0.125 mg/l should be included in laboratory sensitivity tests.

Anti-Infective Agents↗
PubMed1997

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

[Thoracoscopic sympathectomy in the surgical treatment of axillary and palmar hyperhidrosis].

47 patients with axillary palmar hyperhydrosis underwent this surgery. There were 36 women (76.6%) and 11 men (23.4%) among them. The sympathetic trunk has been coagulated on the level between 2d and 4th ribs on both sides. There were no surgical mortality in this group. Nine patients (9.1%) had a pneumothorax, one patient (2.1%) had a subcutaneous emphysema, the other one had pneumonia and one had wound pyosis. In 43 cases the result of the surgery was very good. In 2 cases bilateral relapse and in 2 cases marked compensatory hyperhydrosis were resistered.

Adult↗