Biomedical subjects

J A Hamilton

Publications and source records attributed to J A Hamilton.

At least 145 records · Page 8Linked to original sources

PubMed1993

Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells.

The effect of thrombin on the fibrinolytic potential of human vascular smooth muscle cells (SMC) in culture was studied. SMC of different origin responded to thrombin treatment with a dose and time dependent increase in tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) levels in both cell lysates and conditioned media with maximum effects achieved at 10-20 IU/ml thrombin. PAI-1 antigen levels also increased in the extracellular matrix of thrombin treated SMC. PAI-2 levels in cell lysates of such SMC were not affected by thrombin. The effect was restricted to active thrombin, since DFP-thrombin and thrombin treated with hirudin showed no increasing effect on t-PA and PAI-1 levels in SMC. Enzymatically active thrombin also caused a four-fold increase in specific PAI-1 mRNA and a three-fold increase in t-PA mRNA. Furthermore we demonstrated the presence of high and low affinity binding sites for thrombin on the surface of SMC with a KD = 4.3 x 10(-10)M and 9.0 x 10(4) sites per cell and a KD = 0.6 x 10(-8) M and 5.8 x 10(5) sites per cell respectively. Thrombin could come in contact with SMC in case of vascular injury or following gap formation between endothelial cells. Our data support the idea that besides its known proliferative effect for SMC, thrombin could also modulate their fibrinolytic system.

Antigens↗

PubMed1993

Rheumatoid arthritis: opposing actions of haemopoietic growth factors and slow-acting anti-rheumatic drugs.

The pathogenesis of rheumatoid arthritis and the mode of action of anti-rheumatic drugs are unknown. This hypothesis proposes that haemopoietic growth factors (colony stimulating factors [CSFs]) have an important role in rheumatoid arthritis as regulators of myelopoiesis and as activators of inflammatory leucocytes. It also suggests that slow-acting anti-rheumatic drugs may work by inhibiting myelopoiesis. This opposition to one of the actions of the CSFs would result in fewer inflammatory cells in the inflamed joints.

Anti-Inflammatory Agents↗

PubMed1993

Production of macrophage colony-stimulating factor (M-CSF) by human articular cartilage and chondrocytes. Modulation by interleukin-1 and tumor necrosis factor alpha.

A specific radioimmunoassay was employed to demonstrate that human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively, produce macrophage colony-stimulating factor (M-CSF) in response to stimulation with interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor alpha (TNF alpha) and TNF beta. Optimum doses were 10-100 U/ml for IL-1 (0.06-0.6 nM IL-1 alpha; 0.02-0.2 nM IL-1 beta) and 1-10 nM for TNF alpha. Low levels of M-CSF were observed in the supernatants of nonstimulated cultures while increased levels of M-CSF in response to IL-1 alpha and TNF alpha were detected following 2 h exposure to the cytokines. IL-1 alpha and TNF alpha did not show synergy for the production of M-CSF when both cytokines were added to cultures. Actinomycin D and cycloheximide inhibited both the basal and IL-1 alpha-induced production of M-CSF, suggesting a requirement for de novo RNA and protein synthesis. Cytokine-induced M-CSF production was also inhibited by the antiinflammatory corticosteroid, dexamethasone, but not by the cyclooxygenase inhibitor, indomethacin. The cytokines IL-4, IL-6, platelet-derived growth factor, leukemia inhibitory factor, transforming growth factor-beta and interferons -alpha and -gamma, each had little or no effect on M-CSF levels, while basic fibroblast growth factor, lipopolysaccharide, and retinoic acid were each weak stimuli. We propose that chondrocyte M-CSF production in response to IL-1 and TNF alpha, and the concurrent destruction of cartilage by these cytokines, could provide a mechanism for the chronic nature of rheumatoid disease.

Cartilage, Articular↗

PubMed1993

Interleukin-4 stimulates expression of urokinase-type-plasminogen activator in cultured human foreskin microvascular endothelial cells.

The effect of interleukin-4 (IL-4) on the fibrinolytic system of human microvascular and macrovascular endothelial cells in culture was studied. Only foreskin microvascular endothelial cells (EC) responded to IL-4 treatment with a dose- and time-dependent increase in urokinase-type plasminogen activator (u-PA) (control: 3.0 +/- 0.8 ng/10(5) cells/24 h; 200 U/mL IL-4: 6.7 +/- 0.8 ng/10(5) cells/24 h), whereas human macrovascular EC remained unaffected. A maximum effect was achieved with 200 U/mL IL-4. Little u-PA activity was detected in the conditioned media of human foreskin microvascular EC (HFMEC) treated without and with IL-4 before plasmin treatment (control: 0.03 +/- 0.003 IU/10(5) cells/20 h; 200 U/mL IL-4: 0.09 +/- 0.007 IU/10(5) cells/20 h). These values increased to 0.18 +/- 0.02 IU/10(5) cells/20 h and 0.53 +/- 0.04 IU/10(5) cells/20 h, respectively, after plasmin treatment, indicating that u-PA is released by HFMEC predominantly in its inactive precursor form single-chain u-PA (scu-PA). u-PA activity increased also in the cell lysates of HFMEC up to 2.5-fold after IL-4 treatment. Plasminogen activator inhibitor type-1 (PAI-1) levels produced by HFMEC remained unaffected by IL-4, whereas tissue-type plasminogen activator (t-PA) levels were slightly decreased when HFMEC were treated with IL-4. These findings were also reflected in the specific mRNA levels as determined by Northern blotting. u-PA-specific mRNA increased significantly in HFMEC in the presence of IL-4, whereas t-PA mRNA and PAI-1-specific mRNA in HFMEC and u-PA specific mRNA in human saphenous vein EC (HSVEC) remained unaffected by IL-4 treatment. Our findings suggest a role for IL-4 in the process of angiogenesis, in addition to its known proliferative effect on human microvascular EC, by increasing the fibrinolytic potential of such EC, thereby facilitating extracellular proteolysis and cell migration.

Blotting, Northern↗

PubMed1993

Induction of leukemia inhibitory factor in human synovial fibroblasts by IL-1 and tumor necrosis factor-alpha.

The cytokines, IL-1 alpha and TNF-alpha, induced a dose-dependent production of leukemia inhibitory factor (LIF) in cultured human synovial fibroblast-like cells, as measured by a radioreceptor competition assay. Significant levels of LIF were first detected in cell supernatants between 2 and 4 h after cytokine addition. Increases were also observed in LIF mRNA steady-state expression. Evidence is presented for down-regulation of the IL-1-induced LIF activity by an endogenous cyclo-oxygenase product(s); the glucocorticoid, dexamethasone, lowered the IL-1-induced LIF activity and mRNA expression. A synergistic effect was noted between the actions of IL-1 alpha and TNF-alpha, and between IL-1 and transforming growth factor-beta. IFN-gamma could not induce LIF formation in the synovial cells but inhibited the stimulatory effect of IL-1. These results suggest that cytokine-stimulated synovial fibroblasts may be a major source of intraarticular LIF production in the joints of patients with inflammatory arthritis. Synoviocyte-derived LIF may activate monocyte/macrophages in the lesions and may contribute to the bone changes and certain systemic manifestations in patients with inflammatory joint disease.

Cells, Cultured↗

PubMed1993

The x-ray crystal structure refinements of normal human transthyretin and the amyloidogenic Val-30-->Met variant to 1.7-A resolution.

The x-ray crystal structures of normal human transthyretin (prealbumin) and the amyloidogenic Val-30-Met variant have been refined at 1.7-A resolution to R-values of 0.168 and 0.179, respectively, for 19,882 and 20,362 reflections (Fobs > 2.0 sigma). Standard deviations for stereochemical parameters are 0.018 and 0.022 A for bond distances, 0.030 and 0.038 A for angle distances, and 0.035 and 0.070 A for planar 1-4 distances. The newly refined normal structure shows improvement over the original structure of Blake and Swan (Blake, C. C. F., and Swan, I. D. A. (1971) J. Mol. Biol. 61, 217-224) in stereochemistry and in the conformation of the loop regions. Residues Arg-103, Thr-123, Asn-124, and Pro-125 have now been resolved, and residues 1-9 and 126-127 have been modeled with the aid of simulated annealing refinement. The functional form of transthyretin is a tetramer, having a cylindrical cavity which will bind thyroxine and an exterior binding site for the complex of retinol with retinol-binding protein. The monomer is a beta barrel flattened to become more like a sandwich with residue 30 in the interior. The methionyl for valyl substitution forces the beta sheets of the monomer as much as 1 A apart, resulting in a distortion of the thyroxine-binding cavity, in agreement with the independent observations that the Met-30 variant has low affinity for thyroxine.

Amino Acid Sequence↗

PubMed1993

X-ray crystal structure of the Ala-109-->Thr variant of human transthyretin which produces euthyroid hyperthyroxinemia.

The structure of the Ala-109-->Thr mutation of human transthyretin, a nonamyloidogenic variant with enhanced thyroxine binding, has been determined by x-ray diffraction to a resolution of 1.7 A. The model, including 175 solvent water molecules, has been refined by constrained least squares to an R-value of 0.157. The standard deviations for protein geometry are 0.016 A for bond distances, 0.5 degree for bond angles, 0.031 A for 1-4 distances, and 0.005 A for deviations of planar groups from their least squares plane. The estimated error in protein atomic coordinates is 0.12 A. Residue 109 extends inward between the two beta sheets which form the major component of the monomer, as does the side chain of residue 30 in the amyloidogenic Met-30 variant. Comparison of the Thr-109 structure with that of the normal shows that the extra atoms of the threonine fit into empty space between sheets and make no extensive changes to the molecular conformation. The substitution at 109 causes small local changes in the secondary structure of the A, G, and H strands resulting in a shift of residues 15-17, 108-110, and 117 in each monomer. The thyroxine-binding sites of the Thr-109 and Met-30 variants and of the normal protein are compared, and the results suggest that the variation in affinity for thyroxine between the three proteins may arise from differences in the size of the binding pocket.

Binding Sites↗

PubMed1993

NMR studies of phospholipase C hydrolysis of phosphatidylcholine in model membranes.

Hydrolysis of phospholipids in biological membranes by phospholipase C (PLC) produces an important second messenger molecule, 1,2-diacylglycerol (DAG), that is essential for the activation of protein kinase C (PKC). While the effects of DAG on model membranes have been investigated earlier, studies on physical properties of DAG introduced into phospholipid bilayers by PLC have been lacking. We present an NMR approach for studying structural and kinetic aspects of PLC-mediated hydrolysis of 13 carbonyl-enriched phospholipids in model membranes (small unilamellar vesicles). The product DAG is readily detected by 13C NMR, and its structural properties as well as those of the model membrane can be monitored continuously. PLC hydrolysis was limited to a low proportion of the model membrane by incorporating a small amount of ester phospholipid into a nonhydrolyzable ether-linked phospholipid matrix. Under these conditions, PLC (Bacillus cereus) hydrolyzed only the monolayer of phosphatidylcholine to which it was exposed (the outer monolayer). The 1,2-DAG product remained associated with the membrane bilayer and did not alter bilayer structure in any detectable way. From the chemical shift data, it is inferred that the DAG has an interfacial conformation similar to that of phosphatidylcholine. These results show that DAG could activate PKC by direct interaction with the enzyme rather than by perturbation of the membrane bilayer.

Diglycerides↗

PubMed1993

Cytokine regulation of the synthesis of plasminogen activator inhibitor-2 by human vascular endothelial cells. Comparison with plasminogen activator inhibitor-1 synthesis.

Plasminogen activators are inhibited by plasminogen activator inhibitors-1 (PAI-1) and -2 (PAI-2). We describe the synthesis of PAI-2 by human vascular endothelial cells (EC) cultured from umbilical vein, saphenous vein and foreskin microvasculature in response to interleukin-1 alpha (IL-1 alpha) and tumour necrosis factor alpha (TNF alpha) and compare it with that of PAI-1. Both PAI-2 and PAI-1 were quantitated by ELISAs. PAI-2 was cell-associated while PAI-1 was secreted by EC. IL-1 alpha and TNF alpha increased the synthesis of PAI-2 and PAI-1 by EC in a dose-dependent manner. IL-1 alpha was a stronger stimulus for PAI-2 synthesis than TNF alpha, while both cytokines were equally effective for PAI-1. Northern blot analysis revealed similar changes in mRNA levels to those in antigen levels. PAI-2 synthesis by cytokine-stimulated EC may be important in thrombus formation and inflammation.

Cells, Cultured↗

PubMed1993

Production of leukemia inhibitory factor by human articular chondrocytes and cartilage in response to interleukin-1 and tumor necrosis factor alpha.

OBJECTIVE: To investigate the production of leukemia inhibitory factor (LIF) by human articular chondrocytes and cartilage. METHODS: Chondrocytes and cartilage were cultured with and without added cytokines, and the conditioned media assayed for LIF by a specific radioreceptor competition assay. RESULTS: Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) stimulated LIF production by chondrocyte monolayers and cartilage. LIF was generally not detected in unstimulated cultures. Northern blot analysis showed increased amounts of LIF messenger RNA in IL-1- and TNF alpha-treated chondrocyte cultures. CONCLUSION: Chondrocytes, stimulated by IL-1 and/or TNF alpha, are potential contributors to the elevated levels of LIF observed in the synovial fluids of patients with rheumatoid arthritis and other inflammatory arthritides.

Aged↗

PubMed1993

Effects of macrophage-colony stimulating factor on human monocytes: induction of expression of urokinase-type plasminogen activator, but not of secreted prostaglandin E2, interleukin-6, interleukin-1, or tumor necrosis factor-alpha.

It is often assumed that macrophage-colony stimulating factor (M-CSF) or CSF-1, as well as granulocyte macrophage-CSF (GM-CSF), can induce inflammatory mediator production by monocytes/macrophages. We demonstrate with elutriation-purified human monocytes that, in contrast to lipopolysaccharide, recombinant human CSF-1 does not induce secretion of prostaglandin E2, interleukin-6 (IL-6), IL-1 beta, or tumor necrosis factor alpha, as measured by immunoassay; however, increased urokinase-type plasminogen activator (u-PA) activity in cell lysates and mRNA was observed. Similar results were obtained when the monocytes were treated with recombinant human GM-CSF. Such increased u-PA expression may contribute to the function of CSF-1 at sites of inflammation.

Cells, Cultured↗

PubMed1993

Regulation of plasminogen activator inhibitor-1 levels in human monocytes.

The regulation of PAI-1 synthesis by elutriation-purified human monocytes was studied in vitro and compared to that for PAI-2. PAI-1 formation, as measured by ELISA, was upregulated by TGF-beta (> or = 1 ng/ml) and surprisingly down-regulated by LPS (100 ng/ml), particularly in the presence of TGF-beta; LPS elevated PAI-2 levels (ELISA) while TGF-beta reduced its basal levels and those in LPS-treated cultures. Concomitant changes in mRNA expression occurred. The glucocorticoid dexamethasone (10(-7) M) elevated PAI-1 and acted in concert with TGF-beta in this regard at both the antigen and mRNA levels; interleukin-4 (IL-4) (250 pM) failed to mimic the steroid in its regulation of PAI-1 formation. Since monocyte/macrophage PA activity is likely to be important in tissue remodeling and cell migration at sites of inflammation and in fibrinolysis, it is proposed from these studies that PAI-1, as well as the usually considered PAI-2, may be involved in the negative control of PA activity in this cell type. The synthesis of each PAI appears to be independently regulated.

Cells, Cultured↗

PubMed1993

Reduction of tumor necrosis factor alpha and interleukin-1 beta levels in human synovial tissue by interleukin-4 and glucocorticoid.

The effects of recombinant human interleukin-4 (IL-4) and the glucocorticoid, dexamethasone, on tumor necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) levels in cultures of rheumatoid and osteoarthritic synovial tissue were studied. Low concentrations of IL-4 and dexamethasone suppressed the levels of both cytokines in the supernatants of both types of tissue after stimulation with lipopolysaccharide (LPS); the IL-1 beta and TNF alpha levels were measured by ELISA. It is suggested that it is the monocyte/macrophage in the synovial tissues that is responsive to the inhibitors. It is proposed that glucocorticoids may act on synovial tissue in this manner in vivo and IL-4 may do so if administered intraarticularly.

Arthritis, Rheumatoid↗

PubMed1993

Cytokine regulation of granulocyte-macrophage colony stimulating factor and macrophage colony-stimulating factor production in human arterial smooth muscle cells.

Smooth muscle cells (SMC) are the major cell type found in the walls of large blood vessels and appear to participate in local immune and inflammatory reactions, as well as in certain vascular diseases. We tested whether human arterial SMC can produce in vitro the colony stimulating factors (CSFs), granulocyte macrophage-CSF (GM-CSF) and macrophage CSF (M-CSF). Untreated internal mammary artery and aortic SMC produced no detectable GM-CSF but constitutively made M-CSF, measured by ELISA and radioimmunoassay, respectively. Interleukin-1 (IL-1) and, to a lesser extent, tumor necrosis factor alpha (TNF alpha) stimulated GM-CSF formation within 3 h; mRNA levels also increased particularly in the presence of the protein synthesis inhibitor, cycloheximide. IL-1, TNF alpha and, in addition, interferon-gamma (IFN-gamma) raised the M-CSF levels within 6 h; cycloheximide potentiated the effects of IL-1 and TNF alpha on mRNA levels. These results suggest that cytokine-stimulated human arterial SMC may be a source of the M-CSF found in atherosclerotic lesions. Since monocytes/macrophages can be activated by GM-CSF and M-CSF, while GM-CSF can also affect granulocyte function, SMC may participate in inflammatory reactions and vascular diseases by releasing these cytokines.

Aorta↗

PubMed1993

Regeneration of antidigoxin binding activity in an antibody by changes surrounding the original binding defect.

A panel of antibodies which differ in their L chain structures and which bind to structurally defined haptens, would be useful in investigating L chain structure and function. In a previous study, chain recombinant antibody CR24 (26-10 H, 45-20 lambda) was produced by hybridoma-hybridoma fusion. Although both parental antibodies bound digoxin with high affinity, CR24 lacked detectable digoxin-binding activity. Hybridoma CR24 was subsequently fused with H chain-loss hybridomas in order to produce a panel of antibodies composed of 26-10 H chains and 26-10 "like" L chains. Two antibodies produced were CR260 which demonstrated digoxin-binding activity and CR256 which did not. CR260 and CR256 expressed only one amino acid difference (Pro to Leu at L-96). This difference resulted in the CR256 binding defect. In this report, two new antidigoxin antibodies are described. One, SR2E7, contained the Pro to Leu (L-96) defect, but still bound digoxin. Binding affinities and binding specificity patterns, as well as complete VL DNA sequence and corresponding protein sequence of the new digoxin binding antibody L chains (SR2E7 and SR1C7) are presented. Both kappa L chains are highly homologous to the 26-10 kappa L chain as well as the BALB/c germline gene K5.1. These results suggest that antibodies which are initially defective in binding activity can be cured by changing specific amino acids involved in determining the binding-site structure. Molecular modelling studies of the binding-site region were completed to address L chain structural changes induced by specific amino acid substitutions.

Amino Acid Sequence↗

PubMed1993

Colony stimulating factors, cytokines and monocyte-macrophages--some controversies.

In the literature concerning induction of monocyte-macrophage inflammatory mediators in vitro by colony stimulating factors, IFN-gamma and TGF-beta, there are many conflicting observations; likewise there is disagreement when the regulation of the synthesis of colony stimulating factors by cytokines is examined. Here, John Hamilton attempts to identify unifying concepts from the conflicting data. He goes on to discuss the experimental variables that have led to the current confusion and to assess the significance of the in vitro studies to physiology and pathology.

Animals↗

PubMed1993

Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression.

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.

Breast Neoplasms↗

PubMed1993

Special aspects of neuropsychiatric illness in women: with a focus on depression.

Many depressive disorders predominate in women. Other psychiatric disorders more prevalent in women than men often occur concomitantly with depression. A variety of these disorders show a premenstrual patterning of symptoms and are responsive to antidepressants. Migraine headaches are also more common in women, show a premenstrual patterning, and are responsive to antidepressants. Other neuropsychiatric and endocrine-related disorders predominating in women are discussed in this review. Greater attention to the special aspects of neuropsychiatric disorders in women may improve diagnosis and treatment.

Antidepressive Agents↗