A cysteine-selective fluorescent probe for the cellular detection of cysteine

A series of coumarin fluorophores (1-3), each bearing a double bond conjugated quinoline unit that can undergo a Michael-type reaction with thiol-containing compounds, is reported. These systems, designed to provide so-called turn-on changes in fluorescence response when exposed to thiols, act as fluorescent chemical sensors for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). In the case of 1, selectivity for Cys over Hcy and GSH is observed, both in terms of analyte-induced signal enhancement and response time. On the basis of fluorescence spectroscopic analyses, DFT calculations, and pH dependent studies this substrate selectivity is ascribed to steric interactions between the substituents on the quinolone units present in 1 and the targeted thiols, as well as to the comparatively lower pK_a value of Cys relative to Hcy and GSH. In aqueous solution, probe 1 was found capable of detecting Cys with a detection limit of 10⁻⁷ m. This system was successfully applied to the fluorescence imaging of intracellular Cys in HepG2 cells.     

Characterization of <Emphasis Type="Italic">Taenia solium</Emphasis> cysticerci microsomal glutathione <Emphasis Type="Italic">S</Emphasis>-transferase activity

Glutathione S-transferase activity has been shown to be associated with the microsomal fraction of Taenia solium. Electron microscopy and subcellular enzyme markers indicate the purity of the microsomal fraction that contains the glutathione S-transferase activity. T. solium microsomes were solubilized under conditions used to solubilize integral microsomal proteins. This procedure proved necessary to obtain enzymatic activity. To characterize this parasite enzyme activity, several substrates and inhibitors were used. The optimum activity for microsomal glutathione S-transferase was found to be pH 6.6, with a specific enzyme activity of 0.9, 0.1, 0.067, 0.03, and 0.05 μmol min⁻¹ mg⁻¹ with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 2,4-hexadienal, and trans-2-nonenal, respectively. No activity of glutathione peroxidase was observed. T. solium microsomes had an _app K _{m (GSH)} = 0.161 μM, _app K _{m (CDNB)} = 14.5 μM, and _app V _max of 0.15 and 27.9 μmol min⁻¹ mg⁻¹ for GSH and CDNB, respectively. T. solium microsomes were inhibited by several glutathione S-transferase enzyme inhibitors, and it was possible to establish a simple inhibition system as well as corresponding K _i ’s for each inhibitor. These results indicate that the T. solium microsomal glutathione S-transferase is different from the parasite cytoplasmic enzymes that catalyze similar reactions.     

Glutathione is an essential metabolite required for resistance to oxidative stress in the yeastSaccharomyces cerevisiae

  Glutathione (GSH) is an abundant cellular thiol which has been implicated in numerous cellular processes and in protection against stress caused by xenobiotics, carcinogens and radiation. Our experiments address the requirement for GSH in yeast, and its role in protection against oxidative stress. Mutants which are unable to synthesis GSH due to a gene disruption in GSH 1, encoding the enzyme for the first step in the biosynthesis of GSH, require exogenous GSH for growth under non-stress conditions. Growth can also be restored with reducing agents containing a sulphydryl group, including dithiothreitol, β-mercaptoethanol and cysteine, indicating that GSH is essential only as a reductant during normal cellular processes. In addition, the GSH 1-disruption strain is sensitive to oxidative stress caused by H₂O₂ and tert-butyl hydroperoxide. The requirement for GSH in protection against oxidative stress is analogous to that in higher eukaryotes, but unlike the situation in bacteria where it is dispensable for growth during both normal and oxidative stress conditions. Received: 20 September / 21 October 1995     

Association of recreational physical activity with homocysteine,folate and lipid markers in young women

We assessed the influence of recreational physical activity in young healthy women on homocysteine, a potential risk factor for cardiovascular disease (CVD). Participants were 124 23-year-old normal-weight Italian recreational athletes (performing 8.7 ± 2.46 h week⁻¹ exercise) and 116 controls. Median blood homocysteine, folate and lipid markers did not differ between athletes and controls. Elevated homocysteine levels at CVD risk ≥12.0 and ≥15.0 μmol l⁻¹ were not different between groups. Continuous homocysteine was inversely related to folate (P < 0.001), positively associated with age (P = 0.009) and creatinine (P = 0.033), but not associated with hours of exercise, body mass index, and lipid markers. Women with folate depletion (<3.0 μg l⁻¹) were 4.5-fold more likely to have homocysteine ≥15.0 μmol l⁻¹. Recreational physical exercise does not adversely impact homocysteine levels among young women. Only low folate significantly increases the risk for hyperhomocysteinemia in young women.     

Elevated circulating levels of matrix metalloproteinases MMP-2 and MMP-9 in pseudoxanthoma elasticum patients

Pseudoxanthoma elasticum (PXE) is a rare disorder predominantly affecting the skin, the eyes, and the cardiovascular system. The disease is caused by mutations in the ABCC6 gene and characterized by ectopic calcification and extracellular matrix (ECM) alterations. Matrix metalloproteinases (MMPs) play a pivotal role in the process of ECM remodeling. In the present study, we investigated matrix metalloproteinases MMP-2 and MMP-9 in PXE patients compared to healthy controls. We analyzed the serum concentrations of MMP-2 and MMP-9 in a cohort of 69 German PXE patients and in 69 healthy, age-, and sex-matched control subjects using commercially available ELISA assays. We found elevated concentrations of both MMPs in the sera of PXE patients. MMP-2 levels were significantly higher in patients than controls (231 ± 5.89 vs 202 ± 5.17 ng/ml, p = 0.0002), as were MMP-9 levels (841 ± 65.9 vs 350 ± 30.8 ng/ml, p < 0.0001). Our findings point to an involvement of matrix metalloproteinases in PXE pathology. ECM remodeling in PXE is reflected by elevated levels of circulating MMP-2 and MMP-9. Those MMPs might, therefore, be applicable as serum markers for the matrix-degradative process in PXE.     

Matrix metalloproteinases in type 2 diabetes and non-diabetic controls: effects of short-term and chronic hyperglycaemia

Introduction

The role of matrix metalloproteinases (MMPs) in type 2 diabetes mellitus (DM) is not clear as increased activation of MMPs in the vasculature contrasts with decreased activity of MMPs in the kidneys, contributing to development of nephropathy.

Material and methods

We measured serum MMP-2 and MMP-9 in 22 subjects with type 2 DM age (mean ± SD) 56.7 ±16.8 years, BMI 31.8 ±4.6 kg/m², HbA_1c 8.45 ±1.78% and in 32 controls, age 39.2 ±16.0 years, BMI 35.2 ±8.5 kg/m². In 15 subjects with 2 DM we also measured MMP-2 and MMP-9 at discharge from hospital and after 3 months (n = 8). In controls, MMP-2 and -9 were also measured during 75 g oral glucose tolerance test (OGTT).

Results

Concentrations of MMP-2 and MMP-9 were lower in subjects with type 2 DM (219 ±62 ng/ml vs. 305 ±63 ng/ml and 716 ±469 ng/ml vs. 1285 ±470 ng/ml, for MMP-2 and MMP-9, respectively, p < 0.05). MMP-9 concentrations fell at 120 min of OGTT from 1675 ±372 ng/ml to 1276 ±422 ng/ml (p < 0.05). In diabetic subjects there was a correlation between MMP-9 and HbA_1c (r = 0.51, p< 0.05). In subjects with diabetes there was a fall of HbA_1c from 9.77 ±1.76% to 8.36 ±1.54% (p < 0.01), at three months post-discharge. There was no difference in MMP-2, but there was a fall in MMP-9 at three months post-discharge in comparison to concentrations observed at admission (854 ±560 ng/ml vs. 500 ±235 ng/ml, p= 0.02).

Conclusions

Matrix metalloproteinases in type 2 and MMP-9 concentrations were lower in subjects with 2 DM than in non-diabetic controls. Regulation of MMPs appears to be complex as hyperglycaemia during OGTT results in a decrease in MMP-9, while chronic hyperglycaemia, reflected by HbA_1c, correlates with MMP-9 concentrations in subjects with 2 DM.     

Strenuous endurance training in humans reduces oxidative stress following exhausting exercise

The aim of this study was to evaluate whether high-intensity endurance training would alleviate exercise-induced oxidative stress. Nine untrained male subjects (aged 19–21 years) participated in a 12-week training programme, and performed an acute period of exhausting exercise on a cycle ergometer before and after training. The training programme consisted of running at 80% maximal exercise heart rate for 60 min · day⁻¹, 5 days · week⁻¹ for 12 weeks. Blood samples were collected at rest and immediately after exhausting exercise for measurements of indices of oxidative stress, and antioxidant enzyme activities [superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT)] in the erythrocytes. Maximal oxygen uptake (V˙O_2max) increased significantly (P < 0.001) after training, indicating an improvement in aerobic capacity. A period of exhausting exercise caused an increase (P < 0.01) in the ability to produce neutrophil superoxide anion (O₂ ^•−) both before and after endurance training, but the magnitude of the increase was smaller after training (P < 0.05). There was a significant increase in lipid peroxidation in the erythrocyte membrane, but not in oxidative protein, after exhausting exercise, however training attenuated this effect. At rest, SOD and GPX activities were increased after training. However, there was no evidence that exhausting exercise enhanced the levels of any antioxidant enzyme activity. The CAT activity was unchanged either by training or by exhausting exercise. These results indicate that high-intensity endurance training can elevate antioxidant enzyme activities in erythrocytes, and decrease neutrophil O₂ ^•− production in response to exhausting exercise. Furthermore, this up-regulation in antioxidant defences was accompanied by a reduction in exercise-induced lipid peroxidation in erythrocyte membrane. Accepted: 26 September 2000     

Regulation of Antioxidants and Phase 2 Enzymes by Shear-Induced Reactive Oxygen Species in Endothelial Cells

Exposure of vascular endothelial cells (ECs) to steady laminar shear stress activates the NF-E2-related factor 2 (Nrf2) which binds to the antioxidant response element (ARE) and upregulates the expression of several genes. The onset of shear is known to increase the EC reactive oxygen species (ROS) production, and oxidative stress can activate the ARE. ARE-regulated genes include phase 2 enzymes, such as glutathione-S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), and antioxidants, such as glutathione reductase (GR), glutathione peroxidase (GPx) and catalase. We examined how shear stress affects the antioxidant/phase 2 enzyme activities and whether ROS mediate these effects. ROS production, measured by dichlorofluorescin fluorescence, depended on level and time of shear exposure and EC origin, and was inhibited by either an endothelial nitric oxide synthase (eNOS) inhibitor or a superoxide dismutase (SOD) mimetic and peroxynitrite (ONOO⁻) scavenger. Shear stress (10 dynes/cm², 16 h) significantly increased the NQO1 activity, did not change significantly the glutathione (GSH) content, and significantly decreased the GR, GPx, GST and catalase activities in human umbilical vein ECs. Either eNOS inhibition or superoxide radical (O₂^•−)/ONOO⁻ scavenging differentially modulated the shear effects on enzyme activities suggesting that the intracellular redox status coordinates the shear-induced expression of cytoprotective genes.     

Glutathione-dependent modulation of exhausting exercise-induced changes in neutrophil function of rats

Reduced glutathione (GSH) plays a central role in maintaining an effective synergism between various physiological and exogenous antioxidants. We tested the effects of GSH andN-acetylcysteine (NAC, a pro-GSH clinical drug), intraperitoneal (i.p.) supplementation and GSH deficiency on exercise-induced leucocyte margination and neutrophil oxidative burst activity. GSH, NAC (1g · kg^–1) or placebo saline was i.p. injected (one or eight times) to male rats (n seven per group). The GSH-deficient rats were prepared by i.p. injections ofl-buthionine-[SR]-sulphoximine (BSO, 6 mmol · 1^–1 · kg^–1) twice daily for 4 days. Exercised animals were subjected to treadmill run to exhaustion. Exhausting treadmill exercise significantly decreased peripheral blood leucocyte count in the controls (P < 0.001). Such exercise-associated leucocyte margination was prevented by GSH supplementation. Peripheral blood neutrophil counts were significantly higher (P < 0.02) in the GSH-supplemented groups compared to the placebo control groups. Exercise-induced increase in peripheral blood neutrophil oxidative burst activity as measured by luminol-enhanced chemiluminescence per volume of blood tended to be higher in the GSH-supplemented group (P < 0.10), and lower in the GSH-deficient rats (P < 0.02). In these experiments, for the first time we have shown that GSH supplementation can induce neutrophil mobilization and decrease exercise-induced leucocyte margination, and that exogenous and endogenous GSH can regulate exercise-induced stimulation of the neutrophil oxidative burst.     

Monocyte-derived dendritic cell subpopulations use different types of matrix metalloproteinases inhibited by GM6001

Matrix metalloproteinases (MMPs) are endopeptidases with the potential to cleave extracellular matrix, support tissue renewal and regulate cell migration. Functional activities of MMPs are regulated by tissue inhibitors of MMPs (TIMPs) and disruption of the MMP–TIMP balance has pathological consequences. Here we studied the expression and secretion of MMPs and TIMPs in CD1a⁻ and CD1a⁺ monocyte-derived dendritic cell (DC) subpopulations. Our results showed that monocytes express TIMPs but lack MMPs, whereas upon differentiation to moDCs and in response to activation signals the expression of MMPs is increased and that of TIMPs is decreased. MMP-9 is expressed dominantly in the CD1a⁻ subpopulation, while MMP-12 is preferentially expressed in CD1a⁺ cells. Experiments performed with the synthetic MMP inhibitor GM6001 revealed that this drug efficiently inhibits the migration of moDCs through inactivation of MMPs. We conclude that modulation of MMP activity by GM6001 emerges as a novel approach to manipulate DC migration under inflammatory conditions.     

Breast cancer cells with inhibition of p38<Emphasis Type="Bold">α</Emphasis> have decreased MMP-9 activity and exhibit decreased bone metastasis in mice

p38 belongs to a family of mitogen-activated protein kinases, which transfer extracellular signals into intracellular responses. p38 is also frequently detected in clinical breast cancer specimens, but its role as a prognostic factor is not known. Of the various p38 isoforms, p38β has been shown to mediate the in vitro invasiveness of breast cancer cells through up-regulation of urokinase plasminogen activator (uPA). We studied the role of p38β in breast cancer bone metastases, using dominant negative blockade approach. Human MDA-MB-231 breast cancer clones stably expressing dominant negative p38β (p38/AF) exhibited decreased basal MMP-9 activity. TGF-β ₁-induced MMP-9 activity was also blunted in these clones, as compared with controls in which TGF-β ₁ up-regulated MMP-9 activity. Consistent with these findings, SB202190, a specific p38 inhibitor, also inhibited TGF-β₁-induced MMP-9 activity in parental cells. The p38/AF clones exhibited also reduced uPA production after growth on vitronectin and decreased cell motility, as compared with controls. VEGF production levels in all the studied clones were similar. The p38/AF clone, which had similar in vitro growth rate as the control pcDNA3 clone, formed significantly less bone metastases in a mouse model, as compared with the control clone. In conclusion, inhibition of the p38β pathway results in decreased MMP-9 activity, impaired uPA expression and decreased motility, all of which may contribute to the decreased formation of bone metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biochemical studies on glutathione S-transferase from the bovine filarial worm Setaria digitata

Setaria digitata is a filarial worm of the cattle used as a model system for antifilarial drug screening, due to its similarity to the human filarial parasites Wuchereria bancrofti and Brugia malayi. Since filarial glutathione S-transferase (GST) is a good biochemical target for antifilarial drug development, a study has been undertaken for the biochemical characterization of GST from S. digitata. Cytosolic fraction was separated from the crude S.digitata worm homogenate by ultracentrifugation at 100,000 g and subjected to ammonium sulfate precipitation followed by affinity chromatography using GSH-agarose column. The kinetic parameters K _m and V _max values with respect to GSH were 0.45 mM and 0.105 μmol min⁻¹ mL^-1 respectively. With respect to 1-chloro-2,4-dinitrobenzene, the K _m and V _max values were 1.21 and 0.117 μmol min⁻¹ mL⁻¹ respectively. The effect of temperature and pH on GST enzyme activity was studied. The protein retained its enzyme activity between 0°C and 40°C, beyond which it showed a decreasing tendency, and at 80°C, the activity was lost completely. The enzyme activity was varying with change in pH, and the maximum GST activity was observed at pH 7.5. Gel filtration chromatographic studies indicated that the protein has a native molecular mass of about 54 kDa. The single band of GST subunit appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis was found to have molecular mass of ∼27 kDa. This shows that cytosolic S. digitata GST protein is homodimeric in nature.     

Nitric oxide synthase in bronchial epithelium and nitric oxide metabolites in the lungs of rats with bronchial asthma after fenoterol inhalation

Nitric oxide synthase of the bronchial epithelium and concentrations of nitric oxide metabolites (NO₂ ⁻ and NO₃ ⁻) in bronchoalveolar lavage fluids were measured in rats with bronchial asthma after fenoterol inhalation. It was suggested that nitric oxide-ergic mechanisms can mediate the effects of inhaled β₂-adrenergic agonists. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 176–179, August, 2000     

Nitric oxide synthase in bronchial epithelium and nitric oxide metabolites in the lungs of rats with bronchial asthma after fenoterol inhalation

Nitric oxide synthase of the bronchial epithelium and concentrations of nitric oxide metabolites (NO₂ ⁻ and NO₃ ⁻) in bronchoalveolar lavage fluids were measured in rats with bronchial asthma after fenoterol inhalation. It was suggested that nitric oxide-ergic mechanisms can mediate the effects of inhaled β₂-adrenergic agonists. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 176–179, August, 2000     

N-acetyl cysteine, L-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes

Abstract.   In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and β-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and β-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se- GSH-Px activity can facilitate the detoxification of reactive oxygen species.     

Change in post-exercise vagal reactivation with exercise training and detraining in young men

We studied the effects of aerobic exercise training and detraining in humans on post-exercise vagal reactivation. Ten healthy untrained men trained for 8 weeks using a cycle ergometer [70% of initial maximal oxygen uptake ( ) for 1 h, 3–4 days·week^–1] and then did not exercise for the next 4 weeks. Post-exercise vagal reactivation was evaluated as the time constant of the beat-by-beat decrease in heart rate during the 30 s (t₃₀) immediately following 4 min exercise at 80% of ventilatory threshold (VT). The and the oxygen uptake at VT had significantly increased after the 8 weeks training programme (P<0.0001, P<0.001, respectively). The t₃₀ had shortened after training, and values after 4 weeks and 8 weeks of training were significantly shorter than the initial t₃₀ (P<0.05, P<0.01, respectively). The change in the t₃₀ after 8 weeks of training closely and inversely correlated with the initial t₃₀ (r=–0.965, P<0.0001). The reduced t₃₀ was prolonged significantly after 2 weeks of detraining, and had returned almost to the baseline level after a further 2 weeks of detraining. These results suggest that aerobic exercise training of moderate intensity accelerates post-exercise vagal reactivation, but that the accelerated function regresses within a few weeks of detraining. Electronic Publication     

Increased Oxidative Stress and Altered Levels of Antioxidants in Chronic Obstructive Pulmonary Disease

An imbalance between oxidative stress and antioxidative capacity has been proposed to play an important role in the development and progression of chronic obstructive pulmonary disease. We carried out a study to assess the systemic oxidant-antioxidant status in patients with chronic obstructive pulmonary disease (COPD) and relate it to the severity of disease. We measured a wide range of parameters of oxidant-antioxidant balance in leukocytes, plasma and red cells of 82 patients with COPD and 22 healthy non-smoking controls (HNC). Lung function was measured by spirometry. Staging of COPD was done as per the recommended guidelines. Red cell antioxidative enzyme activities were altered, with glutathione peroxidase (GSH-Px) having lower, superoxide dismutase (SOD) having greater and catalase having similar activity in patients as compared to HNC. In plasma, ferric reducing antioxidant power (FRAP) and total protein sulfhydryls were lower and GSH-Px, lipid peroxides measured as MDA-TBA products, and protein carbonyls were higher in the patients as compared to HNC. Plasma total nitrates and nitrites (NO_x) were similar in the two groups. Superoxide anion (O₂^•−) release from leukocytes upon stimulation with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and total blood glutathione were also higher in patients as compared to HNC. Plasma FRAP had a positive whereas total blood glutathione had a significant negative correlation with the severity of airways obstruction (FEV₁% predicted). Further, comparisons between clinical stages of severity of COPD revealed significant differences in plasma FRAP and total blood glutathione. Our observations suggest there is a systemic oxidant-antioxidant imbalance in the patients with COPD.     

Glioma invasionin vitro: regulation by matrix metalloprotease-2 and protein kinase C

A hallmark of invasive tumors is their ability to effect degradation of the surrounding extracellular matrix (ECM) by the local production of proteolytic enzymes, such as the matrix metalloproteases (MMPs). In this paper, we demonstrate that the invasion of human gliomas is mediated by a 72 kDa MMP, referred to as MMP-2, and provide further evidence that the activity of MMP-2 is regulated by protein kinase C (PKC). The invasiveness of five human glioma cell lines (A172, U87, U118, U251, U563) was assessed in anin vitro invasion assay and was found to correlate with the level of MMP-2 activity (r ² = 0.95); in contrast, the activity of this 72 kDa metalloprotease was barely detectable in non-invasive control glial cells (non-transformed human astrocytes and oligodendrocytes). Treatment with 1,10-phenanthroline, a metalloprotease inhibitor, or with a synthetic dipeptide, containing a blocking sequence (ala-phe) specific for MMPs, resulted in a > 90% reduction in glioma invasion. Furthermore, this MMP-2 activity could be inhibited by the treatment of tumor cells with calphostin C, a specific inhibitor of PKC. Glioma cell lines treated with calphostin C demonstrated a dose-dependent decrease (IC₅₀ = 30 nm) in tumor invasiveness with a concomitant reduction in the activity of the MMP-2. Conversely, treatment of non-invasive control astrocytes with a PKC activator (phorbol ester) led to a corresponding increase in their invasiveness and metalloprotease activity. These findings support the postulate that MMP-2 activity constitutes an important effector of human glioma invasion and that the regulation of this proteolytic activity can be modulated by PKC.Joon Uhm and Nora Dooley contributed equally to this work.