Biological effects and mechanism of action of farnesyl transferase inhibitors.
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Alkyl and Aryl Transferases↗
Biomedical subjects
L Armstrong
Publications and source records attributed to L Armstrong.
Alizarin-beta-D-galactoside: a new substrate for the detection of bacterial beta-galactosidase.
We describe the synthesis of a new substrate for the detection of bacterial beta-galactosidase. This substrate, alizarin-beta-D-galactoside, is readily hydrolysed to release alizarin which complexes with various metal ions to form brightly coloured chelates. A total of 367 strains of Gram-negative bacteria were examined for their ability to hydrolyse three chromogenic substrates: alizarin-beta-D-galactoside (Aliz-gal), cyclohexenoesculetin-beta-D-galactoside (CHE-gal) and 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). A total of 182 strains (49.6%) were found to hydrolyse at least one of the three substrates. All of these 182 strains (100%) hydrolysed Aliz-gal whereas only 170 (93.4%) and 173 (95.1%) hydrolysed CHE-gal and X-gal, respectively. We conclude that alizarin-beta-D-galactoside is a highly sensitive substrate for the demonstration of beta-galactosidase.
Anthraquinones↗
Tumor necrosis factor alpha can contribute to focal loss of cartilage in osteoarthritis.
UNLABELLED: OBJECTIVE; To evaluate the potential for tumor necrosis factor alpha (TNFalpha)-induced focal loss of cartilage in osteoarthritic (OA) knee joints. DESIGN: Fresh cartilage from specified regions of OA joints was immunostained for TNF-receptor (R) bearing chondrocytes. Cartilage explants from the same regions were cultured with or without small amounts of TNFalpha and cumulative GAG release into supernatants measured. Concentrations of TNFalpha, p55 and p75 soluble (s) TNF-R in supernatants from cultured OA and non-arthritic (NA) synovium were measured by ELISA. RESULTS: TNF-R bearing chondrocytes were identified in OA cartilage; more specimens contained p55 TNF-R- than p75 TNF-R-bearing chondrocytes and differences in TNF-R distribution were apparent in cartilage from different regions of the same knees. TNFalpha at 5, 1, 0.5 and 0.25 ng/ml (but not 0.1 ng/ml) significantly increased glycosaminoglycans (GAG) release from cartilage explants in a dose-dependent manner. Variation in susceptibility to TNFalpha was observed in explants from different sites. TNFalpha and p75 sTNF-R, but not p55 sTNF-R, concentrations were significantly higher in OA, as compared with NA, supernatants. A significant correlation between TNFalpha and p75 sTNF-R measurements was apparent only in NA supernatants. CONCLUSIONS: Variations in chondrocyte TNF-R expression occur in OA cartilage in vivo. TNFalpha at concentrations produced by OA synovium in vitro, can degrade cartilage matrix. In most OA supernatants sTNF-R concentrations were insufficient to abrogate the effects of TNFalpha. Thus conditions exist in some OA knees for TNFalpha to contribute to focal loss of cartilage.
Aged↗
Evaluation of p-naphtholbenzein-beta-D-galactoside as a substrate for bacterial beta-galactosidase.
We describe the synthesis of a new substrate for the detection of beta-galactosidase and evaluate its performance in comparison with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) and cyclohexenoesculetinbeta-D-galactoside (CHE-Gal). Of 206 Enterobacteriaceae strains able to hydrolyze X-Gal, 194 (94.2%) hydrolyzed CHE-Gal and 192 (93.2%) hydrolyzed p-naphtholbenzein-beta-D-galactoside (PNB-Gal). We conclude that PNB-Gal is an effective substrate for the detection of beta-galactosidase.
Chromogenic Compounds↗
Reduced production of interleukin 12 by interferon gamma primed alveolar macrophages from atopic asthmatic subjects.
BACKGROUND: Asthma is characterised pathologically by an inflammatory pulmonary infiltrate rich in T helper (Th) 2 cells and eosinophils. Interleukin (IL)-12 is a heterodimeric cytokine critical for driving the development of uncommitted Th cells to express a Th 1 phenotype. Reduced pulmonary production of IL-12 may therefore play a role in the pathogenesis of asthma by contributing to the pulmonary cytokine imbalance seen in asthma. METHODS: IL-12 p70 protein levels in bronchoalveolar lavage fluid and p70 protein levels and IL-12 messenger RNA in alveolar macrophage cultures from normal and atopic asthmatic subjects were measured. RESULTS: There was a significant difference between the mean IL-12 p70 protein level in the bronchoalveolar lavage fluid from asthmatic subjects (37.5 pg/ml) and from normal subjects (131 pg/ml, p = 0.04). Alveolar macrophages from asthmatic subjects produced significantly less IL-12 protein (30 pg/ml) and messenger RNA than those from normal subjects (69.5 pg/ml, p<0.005). These differences were not caused by inhibition of IL-12 production by IL-10 nor to generalised hyporesponsiveness of asthmatic alveolar macrophages from subjects to the effects of interferon (IFN)-gamma. CONCLUSIONS: Pulmonary IL-12 production is lower in asthmatic subjects. This reduction is not the result of generalised hyporesponsiveness to IFN-gamma. Reduced IL-12 levels may contribute to the development of asthmatic pulmonary inflammation through dysregulation of Th cell development.
Adult↗
Increased expression of functionally active membrane-associated tumor necrosis factor in acute respiratory distress syndrome.
Membrane-associated tumor necrosis factor (mTNF) has recently been shown to induce inflammatory cellular responses previously attributed to the soluble form. The present study measures for the first time the expression and function of mTNF on the surface of alveolar macrophages (AMs) to determine whether it is associated with the development of acute respiratory distress syndrome (ARDS). TNF expression was determined by flow cytometry, and the function of mTNF on the surface of AMs was determined by an in vitro cytotoxicity assay. Tumor necrosis factor (TNF)-alpha bioactivity was measured by bioassay. Soluble TNF receptor (TNFR) protein and messenger RNA (mRNA) expression were measured by enzyme-linked immunosorbent assay and reverse transcriptase/polymerase chain reaction, respectively. Increased detection of mTNF was observed on the surface of AMs derived from subjects with ARDS (mean percentage increase in fluorescence 22.30 +/- 3.50% for subjects with ARDS compared with 7.09 +/- 1.70% for At Risk subjects [P < 0.003]). mTNF cytotoxicity in the bioassay positively correlated with the mTNF expression determined by flow cytometry (r(2) = 0.97). Although there was increased mTNF expression and cytotoxic function in ARDS, there was no significant increase in soluble TNF expression in the bronchoalveolar lavage fluid or the AM supernatants. Lower levels of CD120b-soluble TNFR were detected in the AM supernatants derived from subjects with ARDS compared with At Risk (mean 0.264 +/- 0.058 versus 0.593 +/- 0.143 ng/ml, respectively [P < 0.05]). By contrast, there was increased CD120b mRNA expression in AMs derived from subjects with ARDS (P < 0.03), suggesting that increased surface expression of this receptor may be important in mediating the signal of mTNF. These data demonstrate for the first time the presence of functionally active mTNF on the surface of AMs in ARDS and highlight a potential mechanism for TNF-mediated lung injury.
Acute Disease↗
Targeting integrins alpha v beta 3 and alpha v beta 5 for blocking tumor-induced angiogenesis.
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Animals↗
Integrin alpha(v)beta(3)-mediated activation of apoptosis.
The alpha(v)beta(3) integrin mediates endothelial cell binding to the extracellular matrix and transduces an intracellular signal promoting survival of endothelial cells and various tumor cells. While the alpha(v)beta(3) integrin-mediated survival signal has been shown to be adhesion dependent, a thorough analysis has not been performed comparing the biochemical effects of antagonist binding to alpha(v)beta(3) integrin with the effects induced by the growth of cells in suspension. In this study we demonstrate that expression of alpha(v)beta(3) integrin in human embryonic kidney 293 cells transfers the alpha(v)beta(3) integrin survival pathway to an epithelial cell line. Furthermore, we show that alpha(v)beta(3) integrin-expressing cells respond differently to alpha(v)beta(3) integrin-specific antagonist treatment and growth in suspension conditions. Treatment with the alpha(v)beta(3) antagonist echistatin resulted in an apoptotic response occurring prior to cell detachment and was not observed in either suspended cells or antagonist-treated suspended cells. These data suggest that the death induced by antagonist binding to alpha(v)beta(3) integrin results in an apoptotic signal with different kinetics than the apoptotic signal induced by matrix detachment (anoikis). Since aberrant alpha(v)beta(3) integrin expression in tumor models is thought to play a role in tumor cell survival, these data have implications for the use of alpha(v)beta(3) antagonists as anti-tumor agents.
Annexin A5↗
Inter-relationship between tumour necrosis factor-alpha (TNF-alpha) and TNF soluble receptors in pulmonary sarcoidosis.
BACKGROUND: The importance of tumour necrosis factor-alpha (TNF-alpha) in the pathogenesis of pulmonary sarcoidosis has remained uncertain because of the paucity of clinical features associated with excessive levels of this cytokine. Increased levels of soluble TNF receptors (TNF-R), which are known to inhibit TNF-alpha activity, were recently described in the lungs of subjects with sarcoidosis. We hypothesised that TNF-alpha bioactivity may be inhibited in sarcoidosis by the presence of TNF-R. A study was therefore undertaken to investigate for the first time the relationship between soluble receptors and TNF-alpha bioactivity in the lungs of subjects with sarcoidosis. METHODS: Alveolar macrophages (AMs) from 16 subjects with histologically proven sarcoidosis and 13 healthy controls were cultured in the presence and absence of lipopolysaccharide (LPS). The subjects with sarcoidosis were grouped by radiological assessment into stage I (n = 6) and stage II/III (n = 10). The cell culture supernatants and bronchoalveolar lavage (BAL) fluid were assayed for TNF bioactivity using the WEHI 164 clone 13 assay. Immunoreactive (bound and free) TNF-alpha and free TNF-Rs (p55 and p75) were determined by ELISA. RESULTS: Bioactive TNF-alpha was undetectable in the BAL fluid of all the subjects with sarcoidosis and most of the healthy controls. However, there was significantly more immunoreactive TNF-alpha in the BAL fluid from subjects with sarcoidosis than from the controls (median values 0.304 ng/ml and 0.004 ng/ml, respectively, 95% CI 0. 076 to 0.455, p<0.001). The levels of both p55 and p75 in the BAL fluid were higher in both sarcoidosis groups than in the controls (p<0.0005 and p<0.001, respectively). In LPS stimulated AM supernatants reduced TNF-alpha bioactivity was seen in subjects with stage I sarcoidosis compared with those with stage II/III disease and healthy controls (median 0.333 ng/ml vs 1.362 ng/ml and 2.385 ng/ml, respectively, p<0.01). This contrasted with increased p55 levels in the AM supernatants derived from subjects with stage I sarcoidosis compared with those with stage II/III disease and healthy controls (median 0.449 ng/ml vs 0.058 ng/ml and 0.078 ng/ml, respectively, p<0.01). The levels of p75 were increased in unstimulated AM cultures in subjects with stage II/III disease compared with those with stage I disease and healthy controls (median 0.326 ng/ml vs 0.064 ng/ml and 0.102 ng/ml, p<0.05). CONCLUSIONS: These results indicate that TNF-alpha bioactivity may be inhibited by increased soluble TNF-R in the lungs of subjects with sarcoidosis, and this inhibition may be greater in patients with stage I sarcoidosis than in those with stage II/III disease. This may represent a homeostatic mechanism which protects the lung from excessive TNF production characteristic of chronic inflammation.
Bronchoalveolar Lavage Fluid↗
Vascular endothelial growth factor (VEGF) in inflammatory and malignant pleural effusions.
BACKGROUND: Investigation and management of pleural effusions is an important clinical problem yet the pathogenesis of pleural fluid accumulation is poorly understood. Vascular endothelial growth factor (VEGF) is a potent inducer of capillary permeability that is produced by both malignant and inflammatory cells. A study was undertaken to determine whether VEGF has a potential pathogenic role in the development of pleural effusions and whether VEGF receptors are present on human pleural mesothelial cells. METHODS: Normal and inflamed pleura were examined immunohistochemically for the presence of FLT-1 (the fms-like tyrosine kinase receptor of VEGF). VEGF levels were measured by ELISA in 78 consecutive patients presenting with undiagnosed unilateral pleural effusions and the levels were correlated with the aetiology of the effusions. RESULTS: Immunohistochemical staining of normal and diseased pleura demonstrated the presence of the FLT-1 VEGF receptor on human mesothelial cells. Median VEGF levels were 2500 pg/ml in the malignant group and 305 pg/ml in the non-malignant group (median difference 1397.5 pg/ml (95% CI 851 to 2693), p<0.005). Median VEGF levels varied according to tumour histology. VEGF levels were also significantly raised compared with transudates (median 36.5 pg/ml) in empyema (4651 pg/ml (95% CI 833 to 10 000), p<0.001) and parainfectious effusions (360 pg/ml (95% CI 46 to 597), p<0.005). CONCLUSIONS: This first report of VEGF receptors on pleural mesothelial cells has indicated a potential mechanism for the biological activity of VEGF on pleural tissue. VEGF levels are raised in the majority of exudative effusions, implying a pathogenic role for this molecule in the development of pleural effusions.
Adult↗
Changes in collagen turnover in early acute respiratory distress syndrome.
Pulmonary fibrosis is a well-recognized feature of acute respiratory distress syndrome (ARDS). Using immunoassays of bronchoalveolar lavage (BAL), fluid we investigated the synthesis of type I procollagen (PICP) and type I/II collagen degradation products (COL2-3/4C(short) neoepitope) in patients with ARDS, acute lung injury (ALI), subjects with risk factors for ARDS (At Risk), and healthy/ventilated control subjects. PICP was measured by ELISA as a marker of type I procollagen synthesis. COL2-3/4C(short) neoepitope was measured by an inhibition ELISA as a marker of collagenase degradation of type I/II collagen. BAL was performed initially within 48 h of ventilation (Day 1) and then subsequently on Day 4. Dilution of epithelial lining fluid (ELF) was corrected for by plasma urea comparison. Increased PICP levels were observed in the ELF from ARDS and ALI subjects on Day 1 compared with subjects At Risk (median values, 124.9 and 95.0 ng/ml versus 38.0 ng/ml, respectively, p < 0.0005). By contrast, the levels of COL2-3/4C(short) neoepitope were significantly reduced in the subjects with ARDS versus the At Risk subjects (13.22 ng/ml versus 32.33 ng/ml, p < 0.0005). This translated into a greatly increased PICP:COL2-3/4C(short) ratio in the subjects with ARDS (p < 0.0001). There was a significant decline in the PICP level in the subjects with ARDS between Days 1 and 4 (n = 15, p < 0.05). Linear regression analysis showed a significant association between PICP and lung injury score in the subjects with ARDS (p = 0.01). Our data suggests an early shift in balance between type I collagen synthesis and degradation by collagenase. The resultant increase in type I collagen would favor matrix deposition and the development of pulmonary fibrosis in the lungs of subjects with ARDS.
Adolescent↗
Haptic perception of linear extent.
The perception of linear extent in haptic touch appears to be anisotropic, in that haptically perceived extents can depend on the spatial orientation and location of the object and, thus, on the direction of exploratory motion. Experiments 1 and 2 quantified how the haptic perception of linear extent depended on the type of motion (radial or tangential to the body) when subjects explored different stimulus objects (raised lines or solid blocks) varying in length and in relative spatial location. Relatively narrow, shallow, raised lines were judged to be longer, by magnitude estimation, than solid blocks. Consistent with earlier reports, stimuli explored with radial arm motions were judged to be longer than identical stimuli explored with tangential motions; this difference did not depend consistently on the lateral position of the stimulus object, the direction of movement (toward or away from the body), or the distance of the hand from the body but did depend slightly on the angular position of the shoulder. Experiment 3 showed that the radial-tangential effect could be explained by temporal differences in exploratory movements, implying that the apparent anisotropy is not intrinsic to the structure of haptic space.
Adolescent↗
Construction of a family of biphenyl combinatorial libraries: structure-activity studies utilizing libraries of mixtures.
A set of biphenyl aminoacid building blocks has been synthesized. These were used to construct partially-peptidic combinatorial libraries as equimolar multi-component samples. Activity of members of this library as vitronectin receptor antagonists is described, together with SAR studies of the most active members. These studies illustrate several important features of combinatorial libraries.
Biphenyl Compounds↗
Chloramine T-induced structural and biochemical changes in echistatin.
Echistatin is a member of the disintegrin family of peptides and a potent inhibitor of platelet aggregation and cell adhesion. Echistatin binds to integrin alpha(v)beta3 and alpha(IIb)beta3 receptors with high affinity. Binding is mediated by an RGD-containing loop maintained in an appropriate conformation by disulfide bridges. In this study, we have compared the binding characteristics of echistatin iodinated by either lactoperoxidase or chloramine T method. We show that echistatin labeled by lactoperoxidase method binds to integrin alpha(v)beta3 receptor with high affinity and in a non-dissociable manner very similar to native echistatin. In contrast, chloramine T-labeled echistatin can rapidly dissociate from the receptor. We demonstrate that chloramine T reaction results in the addition of an extra oxygen to the methionine residue adjacent to the RGD motif in echistatin. Modeling studies and molecular dynamic simulation studies show that the extra oxygen atom on the methionine residue can form hydrogen bonds with the glycine and aspartic acid residues of the RGD motif. These structural changes in echistatin help explain the changes in the binding characteristics of the molecule following chloramine T reaction.
Chloramines↗
Human smooth muscle alpha-actin gene is a transcriptional target of the p53 tumor suppressor protein.
Smooth muscle (sm) alpha-actin is expressed in vascular smooth muscle cells and fibroblast cells. Its expression is regulated by cell proliferation and repressed during oncogenic transformation. In this study, we demonstrate that p53 activation is associated with a dramatic increase in organized microfilament bundles and an increase in sm alpha-actin mRNA level. Wild-type p53, but not mutant p53, strongly stimulated human sm alpha-actin promoter activity in p53 null cell lines. The sequences homologous to the p53 consensus sequence and to the p53 binding sequence from the muscle creatine kinase, were found within a specific region of the sm alpha-actin promoter. This sequence was sufficient to confer p53-dependent activation to a heterologous promoter and p53 was capable of binding to this sequence as assessed by gel shift analysis. Ionizing irradiation of colorectal tumor cells caused an increase in alpha-actin mRNA level in a p53-dependent manner. Taken together, these results demonstrate that human sm alpha-actin gene is a transcriptional target for p53 tumor suppressor protein and represents the first example of a cytoskeletal gene with a functionally defined p53 response element.
Actins↗
Mutation analysis of patients with Hermansky-Pudlak syndrome: a frameshift hot spot in the HPS gene and apparent locus heterogeneity.
Hermansky-Pudlak syndrome (HPS) is a rare, autosomal recessive disorder in which oculocutaneous albinism, bleeding, and lysosomal ceroid storage result from defects of multiple cytoplasmic organelles-melanosomes, platelet-dense granules, and lysosomes. As reported elsewhere, we mapped the human HPS gene to chromosome segment 10q23, positionally cloned the gene, and identified three pathologic mutations of the gene, in patients from Puerto Rico, Japan, and Europe. Here, we describe mutation analysis of 44 unrelated Puerto Rican and 24 unrelated non-Puerto Rican HPS patients. A 16-bp frameshift duplication, the result of an apparent founder effect, is nearly ubiquitous among Puerto Rican patients. A frameshift at codon 322 may be the most frequent HPS mutation in Europeans. We also describe six novel HPS mutations: a 5' splice-junction mutation of IVS5, three frameshifts, a nonsense mutation, and a one-codon in-frame deletion. These mutations define an apparent frameshift hot spot at codons 321-322. Overall, however, we detected mutations in the HPS gene in only about half of non-Puerto Rican patients, and we present evidence that suggests locus heterogeneity for HPS.
Albinism, Oculocutaneous↗
Production of interleukin 13 by alveolar macrophages from normal and fibrotic lung.
Human interleukin 13 (IL-13) is a cytokine that has a profound effect on primary immune cells by inducing immunoglobulin production, proliferation of B cells, and the differentiation of cells of the monocytic lineage. IL-13 can inhibit the production of inflammatory cytokines by both macrophages and monocytes. Previously, IL-13 expression has been reported only in cells of the T-cell lineage and the mast cell line HMC-1. We now report the presence of IL-13 mRNA and protein in human alveolar macrophages (AMs) analyzed by the reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunoabsorbent assay (ELISA), respectively, and IL-13 protein in bronchoalveolar lavage fluid (BALF) of subjects with pulmonary fibrosis. We have investigated 13 patients from 49 to 75 yr of age with forms of pulmonary fibrosis, and eight healthy volunteers from 24 to 61 yr of age. Their AMs were obtained by bronchoalveolar lavage (BAL) and purified by adherence. The proportion of BAL purified AMs expressing IL-13 mRNA was increased in those subjects with fibrotic lung disease, in comparison with those from control subjects (11 of 13 versus 2 of 8, P < 0.01). IL-13 protein was detectable in the BALF of 8 of 13 patients with pulmonary fibrosis, but in none of the control subjects. AMs of four subjects with systemic sclerosis were cultured and IL-13 protein was increased in the culture supernatants when compared to the control subjects, although this did not reach significance. These findings show that IL-13 mRNA is not only a product of T cells, but is also expressed in both normal AMs and those from subjects with pulmonary fibrosis, and that at least some of the IL-13 mRNA is translated into protein and secreted in subjects with pulmonary fibrosis. We hypothesize that IL-13 may be expressed by normal human AMs as part of the homeostatic control process but its production may be increased in the presence of inflammatory lung disease.
Adult↗
Note: cyclohexenoesculetin-beta-D-glucoside: a new substrate for the detection of bacterial beta-D-glucosidase.
A new substrate for the detection of bacterial beta-D-glucosidase was evaluated as an alternative to aesculin. This substrate, 3,4-cyclohexenoesculetin-7-beta-D-glucoside, was compared with aesculin for the detection of beta-D-glucosidase in 150 enterococci, 40 streptococci, 12 Listeria sp. and 250 strains of Enterobacteriaceae. In the Gram-positive strains tested, aesculin hydrolysis correlated with hydrolysis of 3,4-cyclohexenoesculetin-7-beta-D-glucoside. In the Gram-negative strains the new substrate was hydrolysed by all aesculin-positive strains and also by four strains (10%) of Escherichia coli which gave a negative aesculin reaction. 3,4-Cyclohexenoesculetin-7-beta-D-glucoside was shown to be a reliable alternative to aesculin and was shown to have significant advantages over aesculin when incorporated into solid media. This was due to the non-diffusible end product produced by hydrolysis of 3,4-cyclohexenoesculetin-7-beta-D-glucoside in the presence of iron.
Bacteria↗