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Biomedical subjects

J Lunec

Publications and source records attributed to J Lunec.

At least 127 records · Page 7Linked to original sources
PubMed1992

Modulators of free radical activity in diabetes mellitus: role of ascorbic acid.

Free radical mechanisms are increasingly being implicated in the pathogenesis of tissue damage in diabetes. Various sources of free radicals may modulate oxidative stress in diabetes, including non-enzymatic glycosylation of proteins and monosaccharide autooxidation, polyol pathway activity, indirect production of free radicals through cell damage from other causes, and reduced antioxidant reserve. Ascorbic acid, which may be a principal modulator of free radical activity in diabetes, is shown to be consumed, presumably through free radical scavenging, thus preserving levels of other antioxidants such as glutathione.

Animals↗
PubMed1992

The expression of functional MSH receptors on cultured human melanocytes.

Although alpha-MSH increases skin darkening in humans, there are several reports that it fails to have melanogenic effects on human melanocytes in vitro. The purpose of this study was to see whether cultured human melanocytes express MSH receptors. Human melanocytes were grown in the absence of artificial mitogens such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin (CT) and incubated for 2 h at room temperature with increasing amounts of 125I-labelled Nle4DPhe7-alpha-MSH with and without excess cold peptide. Binding was saturable and specific: Scatchard analysis gave a Kd of 4.9 x 10(-11) M and approximately 700 binding sites/cell. Human keratinocytes and fibroblasts showed no specific binding. The addition of 1 mM dibutyryl cAMP to the culture medium caused a 62% increase in MSH binding to human melanocytes. A smaller increase (25%) was seen with 10(-9) M CT while 25 mM TPA caused a 24% decrease. These results show that human melanocytes in culture express MSH receptors and that this expression can be modulated by mitogens.

Animals↗
PubMed1992

The selective protection afforded by ebselen against lipid peroxidation in an ROS-dependent model of inflammation.

The effects of an experimental model of hydrogen-peroxide-induced foot pad oedema on indices of oxidative damage to biomolecules have been investigated. We have demonstrated increased levels of fluorescent protein and lipid peroxides occurring in plasma at 24 and 48 h post-injection. In addition, a decrease in the degree of galactosylation of IgG was observed which kinetically related the degree of inflammation and to the increase in protein autofluorescence (a specific index of oxidative damage). The effects of ebselen, a novel organoselenium compound which protects against oxidative tissue injury in a glutathione-peroxidase-like manner, have also been examined in this model. Pretreatment of animals with a dose of 50 mg/kg ebselen afforded significant and selective protection against lipid peroxidation only. This effect may contribute to the anti-inflammatory effect of this agent in hydroperoxide-linked tissue damage.

Animals↗
PubMed1992

Murine melanoma cell differentiation and melanogenesis induced by poly(ADP-ribose) polymerase inhibitors.

Murine melanoma cells treated with the melanocyte-stimulating hormone (MSH) family of peptides undergo differentiation characterized by enhanced melanogenesis and altered morphology. These effects are mediated via the adenylate cyclase-cAMP pathway leading to activation of protein kinase A (PKA). We have discovered that inhibition of a post-translational modification of chromatin proteins, viz. poly(ADP-ribosylation), also induces melanogenesis and differentiation in these cells. A range of competitive inhibitors (benzamide and its derivatives) of the nuclear enzyme poly(ADP-ribose) polymerase (PADPRP; EC 2.4.2.30) was utilized, and their ability to induce melanogenesis reflected their potency as PADPRP inhibitors. These compounds induced melanogenesis at low doses (20 microM-2 mM) which did not affect cell growth or viability. Induction of melanogenesis was not attributable to inhibition of cyclic nucleotide phosphodiesterase by these compounds. MSH treatment caused a transient rise in cAMP levels (up to 200-fold by 5 min and returning to near basal levels by 5 h). It also stimulated PKA activity up to 5-fold, and the temporal kinetics of this activation mirrored the changes in cAMP levels. In comparison, the PADPRP inhibitors had no effect on either of these processes. These data constitute a novel demonstration of a cAMP-independent mechanism for the induction of melanoma cell differentiation, including melanogenesis.

Adenylyl Cyclases↗
PubMed1992

MSH receptor expression and the relationship to melanogenesis and metastatic activity in B16 melanoma.

In this study we have compared the effects of different pro-opiomelanocortin (POMC) peptides on melanogenesis and metastasis and their relationship to MSH receptor expression in B16F1 melanoma cells. All peptides, apart from beta-endorphin, increased melanogenesis and the order of potency was Nle4DPhe7-alpha-MSH greater than alpha-MSH greater than ACTH[1-39] greater than des-acetyl alpha-MSH greater than ACTH[1-24]. A similar order of potency was found for metastasis, except for ACTH [1-24], which had a relatively greater effect on metastasis. These findings suggest that the effects on melanogenesis and metastasis are mediated via the same receptor. The results of ligand binding studies also indicated the presence of a single receptor with a KD value for Nle4DPhe7-alpha-MSH of 62 +/- 16pM. This was consistent with crosslinking studies using [125I] Nle4DPhe7-alpha-MSH which produced a single 50-55 kD band on analysis by SDS-PAGE. However, the relative binding affinities of the different peptides, measured by displacement of [125I]-Nle4DPhe7-alpha-MSH, did not closely correlate with the relative potencies in stimulating melanogenesis and metastasis. This suggests that receptor activation and the subsequent biological response is not determined solely by binding affinity.

Adrenocorticotropic Hormone↗
PubMed1992

Absence of a specific effect of free radicals on HLA-B27.

The spondylitis associated HLA-B27 epitope includes a characteristic unpaired cysteine at amino acid position 67. On some B27 molecules the thiol (-SH) side chain of this residue seems to be available for chemical interactions. The possibility that free radicals produced during inflammation might specifically affect this group was investigated in this work. Cells bearing HLA-B27 were exposed to free radicals generated by ultraviolet irradiation or hydrogen peroxide, and HLA antigens were then measured by flow cytometry. Binding of monoclonal antibodies to B27 was not affected. These results do not support a specific susceptibility of HLA-B27 to damage by free radicals, despite its apparently vulnerable structure.

4-Chloromercuribenzenesulfonate↗
PubMed1992

An investigation of the relationship between free radical activity and vitamin C metabolism in elderly diabetic subjects with retinopathy.

Abnormalities of both free radical activity and ascorbic acid metabolism have been documented in diabetes, but their biological basis is unclear and their relationship unstudied in any detail. This study was designed to compare changes in antioxidant status and free radical reactions in a group of elderly diabetic patients (with and without retinopathy) with those in a group of age-matched control subjects. No significant differences in thiobarbituric acid (TBA) reactivity, red cell glutathione (GSH) concentrations or diene conjugates (DC) between patients and controls were seen despite significant depletion of ascorbic acid in patients with diabetes, especially in those with retinopathy. The results emphasise the present-day difficulties of measuring free radical activity and demonstrate a marked abnormality in ascorbic acid metabolism in diabetes.

Aged↗
PubMed1992

Role of reactive oxygen species in the generation of fluorescence by glycation.

Fluorescence (excitation 360 nm, emission 454 nm) generation in glycated albumin was investigated. Antioxidants and the metal chelator desferrioxamine (DFX) were used to study the mechanism of fluorescence generation. Delipidation studies, reverse phase chromatography and scanning fluorimetry were performed to examine the nature of this fluorescence. The mechanism of action of aminoguanidine, a compound which has been shown to inhibit the formation of visible fluorescence in proteins in vitro and in vivo was investigated in relation to glycation and by comparison with compounds with structural similarities. We conclude that hydrogen peroxide, metal ions and hydroxyl radicals are involved in fluorescence generation in glycated albumin, which is largely lipid in nature, and arises through glycation, amino acid oxidation and changes in bound lipid. Our results suggest that the action of aminoguanidine is not specifically related to blocking of ketoamine groups on glycated proteins as previously suggested.

Albumins↗
PubMed1992

Captopril inhibits the fluorescence development associated with glycation of proteins.

Albumin and immunoglobulin G (IgG) show increased visible fluorescence in diabetic patients, IgG fluorescence being correlated with the presence of diabetic retinopathy. Captopril, an angiotensin converting enzyme (ACE) inhibitor, has free radical scavenging ability, attributable to its thiol group. We compared the scavenging effect of captopril (at doses between 0.5 and 100 microM) with perindoprilat, enalapril and enalaprilat (ACE inhibitors without scavenging ability) and two thiol-containing compounds, mercaptopropionylglycine (MPG) and N-acetylcysteine (NAC) (scavengers with no effect on ACE). Three systems were used to generate visible fluorescence in albumin and IgG; glycation, exposure to copper/hydrogen peroxide and gamma radiation. All three thiol-containing compounds inhibited fluorescence development in IgG and albumin, when fluorescence was generated by glycation or gamma radiation. Other ACE inhibitors had no effect with IgG. Enalapril and perindoprilat showed less effect than captopril with albumin; enalaprilat had no effect. No compound had any effect on fluorescence generation by copper/hydrogen peroxide. Captopril may have an additional antioxidant effect compared to other ACE inhibitors.

Albumins↗
PubMed1991

Disturbed handling of ascorbic acid in diabetic patients with and without microangiopathy during high dose ascorbate supplementation.

Abnormalities of ascorbic acid metabolism have been reported in experimentally-induced diabetes and in diabetic patients. Ascorbate is a powerful antioxidant, a cofactor in collagen biosynthesis, and affects platelet activation, prostaglandin synthesis and the polyol pathway. This suggests a possible close interrelationship between ascorbic acid metabolism and pathways known to be influenced by diabetes. We determined serum ascorbic acid and its metabolite, dehydroascorbic acid, as indices of antioxidant status, and the ratio, dehydroascorbate/ascorbate, as an index of oxidative stress, in 20 matched diabetic patients with and 20 without microangiopathy and in 22 age-matched control subjects. Each study subject then took ascorbic acid, 1 g daily orally, for six weeks with repeat measurements taken at three and six weeks. At baseline, patients with microangiopathy had lower ascorbic acid concentrations than those without microangiopathy and control subjects (42.1 +/- 19.3 vs 55.6 +/- 20.0, p less than 0.01, vs 82.9 +/- 30.9 mumol/l, p less than 0.001) and elevated dehydroascorbate/ascorbate ratios (0.87 +/- 0.46 vs 0.61 +/- 0.26, p less than 0.01, vs 0.38 +/- 0.14, p less than 0.001). At three weeks, ascorbate concentrations rose in all groups (p less than 0.0001) and was maintained in control subjects (151.5 +/- 56.3 mumol/l), but fell in both diabetic groups by six weeks (p less than 0.01). Dehydroascorbate/ascorbate ratios fell in all groups at three weeks (p less than 0.0001) but rose again in the diabetic groups by six weeks (p less than 0.001) and was unchanged in the control subjects. Dehydroascorbate concentrations rose significantly from baseline in all groups by six weeks of ascorbic acid supplementation (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1991

Phorbol ester and bryostatin effects on growth and the expression of oestrogen responsive and TGF-beta 1 genes in breast tumour cells.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) (10 nM) produce a marked reduction in the growth, measured by thymidine uptake, of MCF-7 cells in full growth medium, but had only a small effect on MDA-MB-231 and T47D cells. Bryostatin alone also inhibited growth but to a lesser extent than seen with TPA. The effect of TPA on MCF-7 cells was partially reversed by bryostatin, added simultaneously or after TPA, suggesting bryostatin does not simply mimic TPA in this system. Even though both are believed to act via effects on protein kinase C, bryostatin appears to act as antagonist to the effect of TPA as well as a partial agonist on its own. When the oestrogen receptor positive MCF-7 and T47D cells were maintained in charcoal stripped serum, the increase in DNA synthesis on stimulation with oestradiol was inhibited with 50 nM TPA in MCF-7 cells but not in T47D cells. The effects of these treatments on the expression of two well characterised oestrogen responsive genes pNR2(pS2) and pNR100 (Cathepsin-D) were examined. Rather than preventing transcription of these oestrogen responsive genes, TPA alone increased pNR2 and pNR100 levels in MCF-7 cells and the combined effect of oestradiol and TPA had a marked synergistic effect in increasing the transcript levels of these genes. In T47D cells pNR2 transcripts were not detected and the increase in pNR100 mRNA levels were not affected by TPA. We conclude that the inhibitory effects of TPA on the growth stimulation of MCF-7 cells by oestradiol was not due to a general inhibition of the expression of oestrogen responsive genes. An alternative possibility examined was that the growth inhibitory effect of TPA on MCF-7 cells might be due to stimulation of TGF-beta 1, acting as an autocrine inhibitory growth factor. Oestradiol treatment of MCF-7 cells reduced the levels of TGF-beta 1 mRNA whereas TPA produced a marked increase. The combined effect of TPA and oestradiol further increased TGF-beta 1 mRNA above the levels seen with TPA alone. Bryostatin had little effect on TGF-beta 1 expression either alone or in combination with oestradiol. These observations are consistent with the hypothesis that the inhibitory effect of TPA on MCF-7 cells may be partly due to autocrine inhibition by TGF-beta 1.

Antineoplastic Agents↗
PubMed1991

Oxidative damage to lipids within the inflamed human joint provides evidence of radical-mediated hypoxic-reperfusion injury.

Previous work has established the existence of a pathophysiological environment within the inflamed human joint, capable of sustaining a hypoxic-reperfusion event. Using four different assay systems (two standard and two novel) applied to synovial fluid for the assessment of lipid peroxidation, a series of studies demonstrate that exercise of the inflamed human knee promotes radical-mediated lipid peroxidation within the joint. The implication for novel antioxidant therapeutic approaches to inflammatory joint disease is discussed.

Adult↗
PubMed1991

Chain breaking antioxidant status in rheumatoid arthritis: clinical and laboratory correlates.

The ability of fresh sera to resist attack by peroxyl radicals (TRAP) was found to be significantly lower in 20 patients with rheumatoid arthritis (RA) than in 20 healthy controls, consistent with the existence of a redox stress in RA imposed by inflammation. TRAP values in RA varied inversely with a combination of visual analogue pain scale, duration of early morning stiffness, grip strength, and articular index (reflecting inflammatory activity) using multiple linear regression analysis. The concentration of the antioxidant vitamin ascorbic acid was lower in RA plasma and the oxidation-reduction equilibrium of ascorbic acid was disturbed, giving further support to the existence of a redox stress. The major determinant of TRAP in vitro was found to be serum uric acid in RA and serum vitamin E in controls. Serum urate concentration in RA correlated inversely with oxidative changes in serum albumin and IgG. It is suggested that serum urate might have an antioxidant role under certain conditions by limiting free radical induced oxidative changes to protein during inflammation.

Arthritis, Rheumatoid↗
PubMed1991

Comparison of the free radical scavenging activity of captopril versus enalapril: a three month in vivo study in hypertensive diabetic patients.

Lipid peroxides and fluorescent serum proteins, possible markers of free radical activity, are increased in diabetic patients, particularly those with angiopathy. Captopril, an angiotensin converting enzyme (ACE) inhibitor, scavenges free radicals in vitro independently of ACE inhibition. This is probably due to the presence of a sulphydryl group which is not present in other ACE inhibitor drugs. We have compared the effects of captopril and enalapril on free radical activity in thirty-two diabetic subjects with hypertension (BP greater than 160/95 mmHg). After a three week run-in period on no antihypertensive therapy, patients were randomly allocated to receive captopril or enalapril, the dose titrated according to BP response. After three months, BP was well controlled in both groups and glycaemic control unchanged. Both drugs were associated with a reduction of fluorescent IgG (captopril:Baseline [BL] 0.564 vs. 12 weeks [w] 0.428, P less than 0.05, enalapril:BL 0.603 vs. 12w 0.422 P less than 0.05) and thiobarbituric acid reactive material (captopril:BL 2.35 nmol MDA vs. 12w 1.46 nmol, P less than 0.05, enalapril:BL 2.44 nmol vs. 12w 1.72 nmol, P less than 0.01). In contrast to in vitro studies, there was no significant difference between the drugs when used in therapeutic doses, questioning a hypothesised advantage of captopril over enalapril.

Adult↗
PubMed1990

Reactive oxygen species modify human DNA, eliciting a more discriminating antigen for the diagnosis of systemic lupus erythematosus.

During the development of an ELISA to measure anti-DNA antibodies in systemic lupus erythematosus (SLE) sera, native dsDNA was found not to be the most appropriate antigen to use in ELISA assays for differentiating between SLE patients and those with rheumatoid arthritis (RA), a disease also associated with circulating serum anti-DNA antibodies. By modifying the ELISA technique to incorporate human DNA, denatured by reactive oxygen species, to detect anti-DNA antibodies in SLE sera, results consistently showed an increase in antibody binding when compared with the native antigen; no such trend was observed in the comparable group of RA patients. Using this assay serum anti-dsDNA antibody levels were measured in a group of 20 controls, 20 RA patients (10 seropositive and 10 seronegative) and 30 SLE patients (15 with clinically active disease, 15 with inactive disease). A comparison with the standard radioimmunoassay used to measure anti-DNA antibodies for the diagnosis of SLE showed that the ELISA assay using modified DNA performed better than the standard radioimmunoassay offering an improvement in both clinical specificity and sensitivity. The improved method particularly reduced the problem of false-negative results for SLE patients shown clinically to be either mildly active or inactive.

Adult↗