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J A Hamilton

Publications and source records attributed to J A Hamilton.

At least 73 records · Page 4Linked to original sources
PubMed1998

Different pathways of colony-stimulating factor 1 degradation in macrophage populations revealed by wortmannin sensitivity.

Phosphatidylinositol 3'(OH)-kinase (PI 3-kinase) is activated on stimulation of macrophages with colony-stimulating factor 1 (CSF-1). We studied its potential role in the internalization and degradation of CSF-1 and its receptor in two primary populations of murine macrophages, namely bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM). Even though CSF-1 induced PI 3-kinase activity in both BMM and RPM, wortmannin, a potent inhibitor of PI 3-kinase activity, at concentrations that inhibited PI 3-kinase activity by 90% in these cells, had little or no effect on receptor internalization and degradation in either BMM or RPM or on CSF-1 degradation by BMM. Strong (more than 90%) inhibition was, however, observed for CSF-1 degradation by RPM. These findings suggest that both wortmannin-sensitive and wortmannin-insensitive pathways of ligand degradation exist in macrophages and that, although CSF-1 and CSF-1 receptor share the same endocytic pathway initially, they might be targeted to different compartments at later stages of degradation.

Androstadienes↗
PubMed1998

Identification of different lipid phases and calcium phosphate deposits in human carotid artery plaques by MAS NMR spectroscopy.

Accumulation of lipids in atherosclerotic plaques causes progressive narrowing of the arterial lumen, often followed by thrombosis and ischemia. Currently several different methods, most requiring disruption of the plaque, are used to study the physical properties of lipids accumulated in plaques, and lipid composition is typically determined by chemical analysis of completely disrupted plaques. In this study, 13C magic angle spinning NMR spectroscopy (MAS NMR) was used to determine in situ the lipid composition and molecular organization of all lipid phases in human carotid artery plaques (ex vivo). Protocols were developed to observe signals from one lipid phase without interference from other phases. In addition, 31P MAS NMR detected calcification in plaques by the signals from inorganic phosphate complexed to calcium. Together, 13C and 31P MAS NMR comprise a powerful nondisruptive approach for determining the quantity and phase state of components in arterial plaques.

Calcium Phosphates↗
PubMed1998

A 13C nuclear magnetic resonance study of free fatty acid incorporation in acylated lipids in differentiating preadipocytes.

To understand the role of free fatty acid (FFA) incorporation in the accumulation of lipids in the adipocyte and ultimately in the development of obesity, 13C nuclear magnetic resonance was used to study lipid metabolism in differentiating preadipocytes. The incorporation of 13C=O-labeled FFA into cellular lipids in primary cultured rat preadipocytes and 3T3L1 preadipocytes at different stages of differentiation was monitored by the 13C carbonyl chemical shift. Significant incorporation of palmitic acid into phosphatidylcholine in both the alpha and beta acyl chain positions was found in cells at early stages of differentiation. At later differentiation stages or after extended incubation periods, most of the 13C=O signals were found in the triacylglycerol (TG) molecules. Unsaturated 13C=O-labeled acyl chains were detected in the TG molecules when cells were incubated with saturated 13C=O-labeled FFA, indicating that intracellular dehydrogenation had occurred in the 13C=O-labeled palmitoyl chain. By using 13C-labeled methyl myristate as an internal intensity reference, incorporation of 13C FFA into each acyl chain position of the major intracellular lipids was determined quantitatively.

3T3 Cells↗
PubMed1998

Effects of wortmannin and rapamycin on CSF-1-mediated responses in macrophages.

There are differing views regarding the roles of phosphatidylinositol 3-kinases (PI3-kinases) and p70 S6 kinase (p70s6k) in growth factor-induced cellular responses. One approach that is widely employed to investigate these roles is to use the inhibitors, wortmannin and rapamycin, respectively. This approach is used here to study the responses in macrophages to colony stimulating factor-1 (CSF-1). Wortmannin (> or = 30 nM) and rapamycin (> or = 3 nM) both weakly inhibited CSF-1-stimulated DNA synthesis in murine bone marrow-derived macrophages (BMM), suggesting that there are PI3-kinase- and p70s6k-independent pathways required for the onset of S phase; interestingly the combination of the drugs gave dramatic suppression. Inhibition of DNA synthesis by rapamycin on the BMM was much less than that observed with the CSF-1-dependent cell line, BAC1.2F5. In BMM, wortmannin suppressed CSF-1-stimulated increase in p70s6k activity indicating that PI3-kinase activity may lie upstream. In contrast to some other growth factor/cell systems, no evidence was obtained using the inhibitors for the involvement of PI3-kinase or p70s6k in CSF-1-mediated induction of c-fos mRNA expression or Erk-1 activity; in addition, no evidence was found for an involvement in the CSF-1-mediated increase in cyclin D1 expression or STAT activation. The findings reinforce the need to study the signal transduction cascades relevant to each individual growth factor and preferably not in cell lines.

Androstadienes↗
PubMed1998

Mutation and expression analysis of the putative prostate tumour-suppressor gene PTEN.

The chromosomal region 10q23-24 is frequently deleted in a number of tumour types, including prostate adenocarcinoma and glioma. A candidate tumour-suppressor gene at 10q23.3, designated PTENor MMAC1, with putative actin-binding and tyrosine phosphatase domains has recently been described. Mutations in PTEN have been identified in cell lines derived from gliomas, melanomas and prostate tumours and from a number of tumour specimens derived from glial, breast, endometrial and kidney tissue. Germline mutations in PTEN appear to be responsible for Cowden disease. We identified five PTEN mutations in 37 primary prostatic tumours analysed and found that 70% of tumours showed loss or alteration of at least one PTEN allele, supporting the evidence for PTEN involvement in prostate tumour progression. We raised antisera to a peptide from PTEN and showed that reactivity occurs in numerous small cytoplasmic organelles and that the protein is commonly expressed in a variety of cell types. Northern blot analysis revealed multiple RNA species; some arise as a result of alternative polyadenylation sites, but others may be due to alternative splicing.

Amino Acid Sequence↗
PubMed1998

Concentrations of placental protein 14 in uterine flushings from infertile women: validation of the collection technique and method of expression of results.

Concentrations of various proteins in uterine flushings have been described as a direct method for assessment of the secretory activity of the endometrium. We investigated levels of the endometrial protein known as placental protein 14 (PP14) in flushings obtained from 271 infertile women. Under transvaginal ultrasonographic control, 2 ml of 0.154 M sodium chloride solution were injected into the uterine cavity and re-aspirated, five times. In contrast to previous studies the recovered volume of each flushing was not consistent (range: 0.05-2.1 ml); the volume varied significantly between serial samples obtained from an individual (P = 0.02, one-way ANOVA), different cycle days (P < 0.0001, one-way ANOVA) and women with bilaterally blocked versus patent Fallopian tubes (P < 0.05, Student's t-test). Concentrations of PP14 showed a better correlation with protein content (r = 0.506, P < 0.0001) than with the recovered volume (r = 0.087, P = 0.095). We therefore corrected PP14 concentrations for total protein content as an indicator of the efficiency of the flushing process. Corrected PP14 concentrations varied significantly relative to time since the onset of menstruation (P = 0.001, Kruskal Wallis ANOVA) with higher levels on days 1-8, as previously observed in plasma samples. No significant difference in PP14 levels was found with different causes of infertility. This study shows that uterine flushing is not a consistent process in women with differing physical characteristics and at varying times throughout the menstrual cycle.

Adult↗
PubMed1998

Evaluation of the performance of hysterosalpingo contrast sonography in 500 consecutive, unselected, infertile women.

The performance of hysterosalpingo contrast sonography (Hy Co Sy) as a first-line, outpatient investigation of tubal patency was examined in 500 consecutive, infertile women, at one centre. Hy Co Sy was completed in 463 (92.6%) cases, using a galactose microbubble contrast agent (Echovist-200) and transvaginal sonography. Initial plain scanning identified adnexal pathology in 198 women (39.6%). Examination with Echovist was attempted for 905 tubes and only 67 (7.4%) were not assessable; after the first 100 women this decreased to 35 tubes (4.8%). A sonographic appearance compatible with blocked tubes was found on 118 (14.1%) occasions but it was also possible to identify variations in the appearance/filling/spilling patterns of individual tubes which increased the number assessed as abnormal to 193 (23.0%). Comparison with laparoscopy and dye chromopertubation findings from the past three years was possible for 185 (37%) women, representing 282 tubes, which gave Hy Co Sy an overall concordance rate of 85.8%, sensitivity of 90.4%, specificity of 70.3%, positive predictive value of 91.2% and negative predictive value of 68.2%. Some 51.0% of women described only mild discomfort and there were no significant postprocedure complications. Hy Co Sy appears to be an acceptable first-line screen and may select out women in whom more invasive investigations are likely to reveal pathology.

Adult↗
PubMed1998

Routine use of saline hysterosonography in 500 consecutive, unselected, infertile women.

Saline hysterosonography was attempted as a routine, first-line screening test of uterine structure in 500 consecutive, unselected, infertile women. The procedure was completed in 96.8% (484/500) women and the observations were interpretable in 483 of these women. Intrauterine pathology was suspected in 67/499 (13.4%) women on plain ultrasound scan and 58/484 (12%) women with saline hysterosonography. Ultrasound alone had a superior specificity (96.3%) to sensitivity (81.8%) and better negative (97.6%) than positive (73.8%) predictive value for the detection of any intrauterine abnormality, using saline hysterosonography as the reference procedure. Suspected pathology at saline hysterosonography led to hysteroscopy in 20 women, after a median of 5.7 months (range, 1-14). The overall concordance rate between the two procedures was 65% with lesions suspicious of intrauterine polyps not present at subsequent hysteroscopy on six occasions. Criteria were established to help identify women with potentially self-limiting lesions, in whom a re-scan should be considered before resorting to hysteroscopy. The procedure was well tolerated with no significant complications. Saline hysterosonography appeared to be an acceptable first-line screening procedure for uterine structure which enhanced the predictive power of ultrasound alone for uterine anomalies and provided additional information which was potentially of value when planning operative hysteroscopy.

Adult↗
PubMed1998

Fatty acid transport: difficult or easy?

Transport of unesterified fatty acids (FA) into cells has been viewed either as a simple diffusion process regulated mainly by lipid physical chemistry or as a more complex process involving protein catalysis. In this review FA transport in cell membranes is broken down into three essential steps: adsorption, transmembrane movement, and desorption. The physical properties of FA in aqueous, membrane, and protein environments relevant to transport mechanisms are discussed, with emphasis on recent information derived from NMR and fluorescence studies. Because of their low solubility in water and high hydrophobicity, FA bind rapidly and avidly to model membranes (phospholipid bilayers); if albumin is a donor, FA desorb rapidly to reach their equilibrium distribution between the membrane and albumin. The ionization properties of FA in a phospholipid bilayer result in a high population of the un-ionized form (approximately 50%) at pH 7.4, which diffuses across the lipid bilayer (flip-flops) rapidly (t(1/2) < 1 sec). Desorption of FA from a phospholipid surface is slower than transmembrane movement and dependent on the FA chain length and unsaturation, but is rapid for typical dietary FA. These physical properties of FA in model systems predict that proteins are not essential for transport of FA through membranes. The only putative FA transport protein to be purified and reconstituted into phospholipid bilayers, the mitochondrial uncoupling protein (UCP1), was shown to transport the FA anion in response to FA flip-flop. New experiments with cells have found that FA movement into cells acidifies the cytosol, as predicted by the flip-flop model.

Adsorption↗
PubMed1997

Fatty acid binding proteins reduce 15-lipoxygenase-induced oxygenation of linoleic acid and arachidonic acid.

Free fatty acids in plasma and cells are mainly bound to membranes and proteins such as albumin and fatty acid binding proteins (FABP), which can regulate their biological activities and metabolic transformations. We have investigated the effect of FABP and albumin on the peroxidation of linoleic acid (18:2) and arachidonic acid (20:4) by 15-lipoxygenase (15-LO). Rabbit reticulocyte 15-LO produced a rapid conversion of [1-14C]18:2 to 13-hydroxyoctadecadienoic acid (13-HODE) and [3H]20:4 to 15-hydroxyeicosatetraenoic acid (15-HETE). 13-HODE formation was reduced when intestinal FABP (I-FABP). liver FABP (L-FABP) or albumin was added. The relative ability of these proteins to reduce 15-LO induced formation of 13-HODE and 15-HETE was BSA > L-FABP > I-FABP. Smaller reductions in activity were observed with 20:4 as compared to 18:2. The IC50-values of I-FABP and L-FABP, using either 18:2 (3.4 microM) or 20:4 (3.4 microM), were 4.6 +/- 0.6 and 1.9 +/- 0.2 microM, respectively, for reduction of 13-HODE and 6.8 +/- 0.3 and 3.1 +/- 0.2 microM, respectively, for reduction of 15-HETE formation. The smaller 15-HETE reduction correlated with decreased binding of 20:4 to the FABP. Titration calorimetry also showed that the I-FABP IC50 for 18:2, 0.25 microM, was lower then for 20:4, 0.6 microM. Thus the reduction in fatty acid lipid peroxidation relates to the binding capacity of each FABP. We also demonstrated that 18:2 rapidly diffuses (flip-flops) across the phospholipid bilayer of small unilamellar vesicles (SUV) and measured partitioning of 18:2 between proteins and SUV by the pyranin fluorescence method [Kamp, F. and Hamilton, J.A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11367-11370]. Addition of proteins to SUV in buffer resulted in a complete desorption of 18:2 from SUV with a relative effect of BSA > L-FABP > I-FABP. This suggests that the relative effects of these proteins on 18:2 peroxidation will not be altered by the presence of membranes. Our results indicate that FAPBs protect intracellular polyunsaturated fatty acids against peroxidation and, through differential binding of 18:2 and 20:4, they may modulate the availability of these polyunsaturated fatty acids to intracellular oxidative pathways.

Animals↗
PubMed1997

cAMP suppresses p21ras and Raf-1 responses but not the Erk-1 response to granulocyte-colony-stimulating factor: possible Raf-1-independent activation of Erk-1.

The cAMP analogue 8-bromo-cAMP (8BrcAMP) inhibits granulocyte-colony-stimulating factor (G-CSF)-stimulated DNA synthesis in myeloid NFS-60 cells. We examined the effect of 8BrcAMP addition on the G-CSF-stimulated extracellular signal-related protein kinase 1 (Erk-1), p21ras and Raf-1 activation. The Erk-1 activity was not down-regulated by the increase in intracellular cAMP levels, whereas p21ras and Raf-1 activities were, suggesting that Erk-1 activity might not be dependent on upstream p21ras and/or Raf-1 activity in this system. To explore this possibility further, we sought to determine whether there were downstream substrates of Raf-1 that were distinguishable from those of Erk-1 by using two-dimensional SDS/PAGE analysis of the protein phosphorylation patterns of NFS-60 cell cytosolic extracts treated with exogenous Raf-1 or Erk-1 in the presence of [gamma-32P]ATP. The two phosphorylation patterns were found to have many differences. To gain further insights into the possible relevance of these phosphorylation patterns and as an approach to exploring in more detail the inhibitory effect of 8BrcAMP, two-dimensional SDS/PAGE analysis was performed on the cytosolic extracts of 32P-labelled NFS-60 cells treated with G-CSF, in the absence or presence of 8BrcAMP. It was found that the phosphorylated proteins whose appearance was specific to the action of exogenous Raf-1 were sensitive to the action of 8BrcAMP in vivo, whereas those whose appearance was specific to the action of exogenous Erk-1 alone, or common to the actions of Raf-1 and Erk-1, were 8BrcAMP-insensitive. The results are consistent with a Raf-1-independent pathway for Erk-1 activation in G-CSF treated myeloid cells, and a number of potential downstream substrates of these kinases have been identified for further characterization.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

CSF-1 and cell cycle control in macrophages.

Control of cell proliferation involves a finely interwoven network of positive and negative cell cycle regulators. Signal transduction pathways linking c-fms (CSF-1R) to cellular proliferation and differentiation are being explored. Part of the strategy is to use a series of G1 inhibitors to help pinpoint relevant targets. Several inhibitors-8Br-cAMP, interferon gamma (IFN gamma), INF alpha/beta, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF alpha), and dimethylamiloride-suppress CSF-1-stimulated proliferation in murine bone marrow-derived macrophages (BMM) even when added in the mid- to late-G1 phase of the cell cycle. The down-modulating effects of the inhibitors on the expression of the following cell cycle regulators have been examined: c-myc, cyclin D1 and D2, cdk4, Rb phosphorylation, E2F binding activity, ribonucleotide reductase subunits, and PCNA. Some differences in the negative control of such regulators were found, for example, in the manner in which IFN gamma and cAMP down-regulate c-myc expression. Using blocking antibodies and BMM from type I IFN receptor knockout mice, it appears that one of these inhibitors, IFN alpha/beta, acts as an endogenous inhibitor in CSF-1-treated BMM and is also responsible, at least in part, for the inhibition of cell cycle progression by LPS and TNF alpha. Another strategy has been to attempt to relate early biochemical changes induced by CSF-1 to later changes in the G1 phase, partly by studying cycling versus noncycling macrophages and partly by using cells expressing c-fms with tyrosine mutations in the intracytoplasmic region. CSF-1-mediated effects on the following signal transduction molecules in these systems will be described: PI3-kinase, myelin basic protein kinases, Erks, and STAT transcription factors.

Animals↗
PubMed1997

CSF-1 signal transduction.

Colony-stimulating factor-1 (CSF-1) or macrophage-CSF (M-CSF) is a growth factor involved in the proliferation, differentiation, and activation of cells of the monocyte/macrophage lineage. Its receptor is the homodimeric, tyrosine kinase product of the c-fms proto-oncogene, which contains a so-called kinase insert domain. This review focuses mainly on recent studies of signal transduction events that are initiated on interaction of CSF-1 and its receptor. A summary is given of the tyrosine autophosphorylation sites on c-Fms identified to date, including their interaction with various substrates and their possible significance for signal transduction and cellular function. In addition, the signal transduction pathways that have been identified to lie downstream of activated c-Fms are reviewed. Although it is apparent that there have been many recent significant developments in our understanding of CSF-1 signaling, a number of examples are mentioned of significant discrepancies in the literature, some possible reasons for which can sometimes be offered. It is also apparent that any particular biochemical response or signal transduction pathway, even though widespread in other ligand receptor/cellular systems, including those with similar receptor structures to c-Fms, may not be relevant to CSF-1 signaling. The relevance of any potentially important molecular signaling pathway activated by CSF-1 in cells in vitro will ultimately have to be related to the functions of monocytes/macrophages in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed1997

G1 phase arrest of human smooth muscle cells by heparin, IL-4 and cAMP is linked to repression of cyclin D1 and cdk2.

Smooth muscle cell proliferation is a key event in the development of atherosclerosis. Inhibition of this proliferation may lead to better prevention and treatment of the disease. While a number of agents have been found to inhibit SMC proliferation, their mechanisms of action are not fully understood. We wanted to determine the effects of three physiologically relevant anti-mitogenic agents on two classes of proteins which have major roles in cellular proliferation, namely cyclins and cyclin-dependent kinases (cdks). Following stimulation with fetal calf serum (FCS), quiescent human umbilical artery smooth muscle cells (HUASMC) synthesised cyclin D1 mRNA and protein and cdk2 mRNA in the G1 phase, whereas cdc2 protein was expressed after the onset of the S phase. Heparin, a strong inhibitor of HUASMC proliferation, strongly down-modulated the levels of cyclin D1 mRNA and protein, cdk2 mRNA and cdc2 protein. Interleukin-4 (IL-4) or 8-bromo-adenosine 3',5'-cyclic monophosphate (cAMP) also lowered the levels of these cell cycle regulatory proteins, although their effects were relatively weak, reflecting their only partial inhibition of HUASMC DNA synthesis. There was specificity in the cell cycle targets of the agents since none appeared to affect the levels of cdk4 protein.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Solution structure of human intestinal fatty acid binding protein: implications for ligand entry and exit.

The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy. Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra (NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel beta-strands which form two nearly orthogonal beta-sheets of five strands each, and two short alpha-helices that connect the beta-strands A and B. The interior of the protein consists of a water-filled cavity between the two beta-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP. The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand.

Amino Acid Sequence↗
PubMed1997

Identification of phosphoproteins specific to granulocyte colony-stimulating factor-mediated signaling using 2D-SDS-PAGE.

Like other cytokines, granulocyte colony-stimulating factor (G-CSF) activates a complex array of signal transduction pathways involving multiple kinases and phosphatases. We sought to identify phosphoproteins specific to G-CSF signaling. Using 2D-SDS-PAGE of 32P-labeled cytosolic extracts, we compared phosphoprotein patterns of NFS-60 cells treated with G-CSF or interleukin-3 (IL-3). We also compared the patterns found after stimulation of M-NFS-60 cells with macrophage-CSF (M-CSF). A large number of phosphoproteins were found that were specific for the G-CSF response. Their distribution contrasted with that of Erk-1-related spots, identified by Western blotting, which were common to G-CSF, M-CSF (CSF-1), and IL-3 responses. The activation of Erk-1 by these cytokines was confirmed by in vitro kinase assays. The 2D-SDS-PAGE approach was also used to demonstrate that a series of unrelated G1 phase inhibitors of the mitogenic action of G-CSF elicited both common and diverse protein phosphorylation changes in G-CSF-treated NFS-60 cells that were not dependent on the inhibition of Erk-1 activity, as demonstrated by both in vitro kinase assays and 2D-SDS-PAGE. Therefore, 2D-SDS-PAGE has potential to dissect both the signal transduction pathways lying downstream of the G-CSF receptor (and of the receptors for other CSFs) and also the site of action of proliferation inhibitors.

Animals↗