Biomedical subjects

Rui Curi

Publications and source records attributed to Rui Curi.

At least 55 records · Page 3Linked to original sources

PubMed2004

The mechanism of indole acetic acid cytotoxicity.

The cytotoxic effect of IAA (1 mM) was examined in rat neutrophils and lymphocytes by: loss of membrane integrity (necrosis), DNA fragmentation, chromatin condensation and mitochondrial transmembrane potential (apoptosis). The following conditions were studied: (1) rat neutrophils (high peroxidase activity; (2) rat lymphocytes in the absence and presence of horseradish peroxidase; and (3) rat lymphocytes co-cultivated with rat neutrophils. Incubation of neutrophils with IAA induced loss of membrane integrity, chromatin condensation and DNA fragmentation. The same was observed in lymphocytes incubated with HRP and IAA or co-cultivated with neutrophils in the presence of IAA. Neutrophils and lymphocytes incubated with IAA presented a more pronounced depolarization of mitochondrial transmembrane potential after 12 h treatment. Incubation for 24 h in the presence of IAA (1 mM) showed increase in the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase. The addition of exogenous antioxidant enzymes (SOD and CAT) prevented the loss of cell membrane integrity induced by IAA. Therefore, the process of cell death induced by IAA involves ROS production.

Animals↗

PubMed2004

Effects of EPA and DHA on proliferation, cytokine production, and gene expression in Raji cells.

The effects of EPA and DHA on the function and gene expression of a B-lymphocyte cell line (Raji) were investigated. Proliferation; production of interleukin-10 (IL-10), tumor necrosis factor (TNF)-alpha, and interferon (INF)-gamma; and expression of pleiotropic genes were evaluated. Cell proliferation was increased in the presence of 12.5 microM EPA (approximately twofold) and 12.5 microM DHA (approximately 1.5-fold). EPA and DHA (25 microM) also decreased production of the key immunoregulatory cytokines IL-10, TNF-alpha, and INF-gamma. EPA and DHA changed the expression of specific genes, but this effect was more marked for EPA (25.9% of genes investigated) compared with DHA (8.4% of genes investigated). EPA and DHA affected the expression of genes clustered as: cytokines, signal transduction, transcription, cell cycle, defense and repair, apoptosis, cell adhesion, cytoskeleton, and hormones. The most remarkable changes were observed in the genes of signal transduction and transcription. These results led us to conclude that the mechanism of DHA and EPA effects on B-lymphocyte functions includes regulation of gene expression. Thus, the ingestion of fish oil, a rich source of EPA and DHA, may have a strong effect on B-lymphocyte function in vivo. However, remarkable differences were observed between DHA and EPA, demonstrating that specific effects of these FA may be responsible for the marked differences in edible oil effects on immune function in vivo reported by others.

Apoptosis↗

PubMed2004

Comparative toxicity of oleic acid and linoleic acid on Jurkat cells.

BACKGROUND: Lipid emulsions for parenteral nutrition commercially available are mainly composed of long-chain triacylglycerol containing a high proportion of alpha-6 polyunsaturated fatty acids or alpha-9 monounsaturated fatty acids. The immunological impact of such therapy is particularly important because parenteral and enteral diets are often administered to critical ill patients. The comparative toxicity of oleic acid and linoleic acid on Jurkat cells, a human T lymphocyte cell line, and the type of cell death induced by these fatty acids were determined. METHODS: Cell death was investigated by cytometry: decrease in cell volume, increase of granularity, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, lipid accumulation; by fluorescence microscopy: chromatin condensation and acridine orange/ethidium bromide assay; and by RT-PCR: mRNA expression of apoptotic genes. RESULTS: Evidence is presented herein that oleic acid is much less toxic to Jurkat cells than linoleic acid. Both fatty acids promote apoptosis and necrosis of these cells. The mechanism of cell death induced by these fatty acids seem to involve with mitochondrial depolarization, lipid accumulation and the levels of C-MYC and P53 mRNA expression. CONCLUSION: Therefore, oleic acid may offer an immunological less harmful alternative to linoleic acid for parenteral and enteral diets preparation.

Apoptosis↗

PubMed2004

Ciglitizone and 15d PGJ2 induce apoptosis in Jurkat and Raji cells.

Several studies have shown that PPARgamma agonists play a role in the regulation of lymphocytes function and apoptosis. However, the molecular mechanism(s) underlying the immunomodulatory effects of PPARgamma agonists are not defined yet. In this study, the effects of PPARgamma (15d PGJ2 and ciglitizone) ligands on proliferation, cytokine production and apoptosis of Jurkat and Raji cells (human T and B lymphocytes, respectively) were examined. Ciglitizone and 15d PGJ2 presented antiproliferative and cytotoxic effects on Jurkat and Raji cells as shown by [14C]-thymidine incorporation and cell viability assay. In addition, 15d PGJ2 inhibited cytokine production (IL-2 in Jurkat cells and IL-10 in Raji cells). The mechanism whereby PPARgamma agonists induced cytotoxicity is via apoptosis as shown by DNA fragmentation, nuclear condensation and phosphatidylserine externalization. The induction of apoptosis by ciglitizone and 15d PGJ2 on Jurkat and Raji cells may explain the suppression of cytokine production and the decrease in proliferation observed in both cell types. The apoptotic process was associated with a decrease in mitochondrial membrane potential and a marked down-regulation of the c-myc expression. These findings might play a key role in the apoptosis of T and B lymphocytes induced by PPARgamma agonists.

Annexin A5↗

PubMed2004

Fish oil supplementation in F1 generation associated with naproxen, clenbuterol, and insulin administration reduce tumor growth and cachexia in Walker 256 tumor-bearing rats.

Weanling female Wistar rats were supplemented with fish oil (1 g/kg body weight) for one generation. The male offspring received the same supplementation until to adult age. Rats supplemented with coconut fat were used as reference. Some rats were inoculated subcutaneously with a suspension (2 x 10(7) cells/mL) of Walker 256 tumor. At day 3, when the tumor was palpable, rats were treated with naproxen (N) (0.1 mg/mL), clenbuterol (Cb) (0.15 mg/kg body weight), and insulin (I) (10 U/kg body weight). At day 14 after tumor inoculation, the animals were killed. Tumor was removed and weighed. Blood, liver, and skeletal muscles were also collected for measurements of metabolites and insulin. In both tumor-bearing untreated rats and tumor-bearing rats supplemented with coconut fat, tumor growth, triacylglycerol, and blood lactate levels were higher, and glycogen content of the liver, blood glucose, cholesterol and HDL-cholesterol levels were lower as compared with the non-tumor-bearing and fish oil supplemented groups. Fish oil supplementation of tumor-bearing rats led to a partial recovery of the glycogen content in the liver and a full reversion of blood glucose, lactate, cholesterol, and HDL-cholesterol levels. The treatment with N plus Cb plus I attenuated cancer cachexia and decreased tumor growth in both coconut fat and fish oil supplemented rats. In conclusion, chronic fish oil supplementation decreased tumor growth and partially recovered cachexia. This beneficial effect of fish oil supplementation was potentiated by treatment with naproxen plus clenbuterol plus insulin.

Animals↗

PubMed2004

Comparative effects of eicosapentaenoic acid and docosahexaenoic acid on proliferation, cytokine production, and pleiotropic gene expression in Jurkat cells.

Comparative effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) acid on Jurkat T cells were investigated. The following parameters were evaluated: concanavalin A (Con A) induced proliferation, production of interleukin-2 (IL-2), IL-4, IL-10, and interferon-gamma (INF-gamma), and expression of pleiotropic genes by macroarray technique (83 genes in total). DHA inhibiting effect on Con A-induced proliferation was more pronounced than that of EPA. The decrease in IL-2 and INF-gamma production was observed for both fatty acids, whereas the production of IL-10 was decreased by EPA only. The expression of a significant proportion of genes was altered by the fatty acids; 30% for DHA (25 genes) and 26.5% for EPA (22 genes). DHA and EPA markedly affected the expression of genes clustered as cytokines and related receptors, signal transduction pathways, transcription factors, cell cycle, defense and repair, apoptosis, DNA synthesis, cell adhesion, cytoskeleton, and hormone receptors. Therefore, the effect of fatty acids on T-lymphocyte function involves regulation of expression of important genes. Marked differences were observed between the effects of EPA and DHA, indicating that it is an over-simplification to generalize the effects of n-3 fatty acids.

Cell Proliferation↗

PubMed2004

Effects on Walker 256 tumour of carmustine associated with a cholesterol-rich microemulsion (LDE).

A cholesterol-rich microemulsion that binds to low-density lipoprotein (LDL) receptors (LDE), after injection into the bloodstream, concentrates in neoplastic tissues that over-express those receptors. LDE can thus serve as a vehicle for drug targeting. It was shown that carmustine side effects are pronouncedly reduced when the drug is associated with LDE in cancer patients. In this study, the therapeutic action of LDE associated with carmustine was compared with that of the non-associated drug in rats implanted with Walker 256 tumour. The toxicity and anti-tumour activity in rats treated with either free carmustine or carmustine associated with LDE and in control rats treated with saline solution were determined after a single intraperitoneal injection. The LD90 (90% lethal dose) of LDE-carmustine was 77 mg kg(-1) and of free carmustine was 44 mg kg(-1), indicating that LDE decreases toxicity. LDE-carmustine was able to decrease tumour mass at a lower dose level than free carmustine. Tumour regression time was shorter in LDE-carmustine- than in free carmustine-treated animals. Therefore, this study shows that the association of carmustine with LDE increases the therapeutic index of carmustine.

Animals↗

PubMed2004

Beneficial effect of glutamine on exercise-induced apoptosis of rat neutrophils.

INTRODUCTION/PURPOSE: The effect of a single bout of intensive exercise on apoptosis of rat neutrophils and the possible prevention by glutamine administration was examined. The experiments were performed in sexually immature and sexually mature male rats as to examine the possible involvement of sexual maturation in the effect of exercise. METHODS: Exercise was carried out on a treadmill for 1 h before rats were killed by decapitation. Aqueous solution of glutamine was given by gavage (1 g.kg-1 body weight), 1 h before exercise. Neutrophils were obtained by intraperitoneal lavage with phosphate-buffered saline (PBS), 4 h after injection of oyster glycogen solution. The cells were then analyzed for apoptosis by flow cytometry and fluorescence microscopy. Pro- and antiapoptotic gene expression was evaluated by reverse transcriptase chain reaction (RT-PCR). RESULTS: Neutrophils obtained from immature and mature exercised rats showed an increase in DNA fragmentation, chromatin condensation, and phosphatidylserine externalization. This suggests that all neutrophils suffered apoptosis. To study the possible mechanism involved, the production of reactive oxygen metabolites, expression of genes involved in apoptosis and mitochondrial transmembrane potential were examined. Acute exercise raised reactive oxygen metabolites production by neutrophils. Exercise did not change the expression of antiapoptotic (bcl-xL) and apoptotic (bax and bcl-xS) genes in neutrophils from immature rats but caused a significant increase of bax and bcl-xS expression and provoked a significant decrease of bcl-xL expression in cells from mature rats. Exercise also induced a marked loss of mitochondrial depolarization in neutrophils. Oral glutamine supplementation partially prevented the exercise-induced apoptosis in neutrophils from sexually immature and mature rats. CONCLUSION: The protective effect of glutamine on neutrophil apoptosis induced by acute exercise possibly occurs by preservation of mitochondrial function.

Administration, Oral↗

PubMed2004

Comparative acute effects of l-carnitine and dl-carnitine on hepatic catabolism of l-alanine and l-glutamine in rats.

AIM: To compare the acute effects of l-carnitine (LCT) and dl-carnitine (DLC) on hepatic catabolism of l-alanine and l-glutamine in rats. METHODS: Livers from 24 h fasted and fed rats were perfused in situ. The substrates l-alanine (5 mmol/L) and l-glutamine (5 mmol/L) were employed. The gluconeogenic and ureogenic activity was measured as the difference between the rates of glucose and urea released during and before the infusion of l-glutamine or l-alanine. RESULTS: LCT (60 micromol/L) but not DLC (60 micromol/L and 120 micromol/L) increased the production of glucose and urea from l-glutamine. However, neither LCT (60 micromol/L and 120 micromol/L) nor DLC (60 micromol/L and 240 micromol/L) showed any significant effect on hepatic glucose and urea production from l-alanine. CONCLUSION: The results showed a different acute effect of LCT and DLC on the activation of hepatic gluconeogenesis and ureagenesis promoted by l-glutamine, reinforcing the idea that DLC could not replace LCT.

Alanine↗

PubMed2003

Effect of arachidonic acid on proliferation, cytokines production and pleiotropic genes expression in Jurkat cells--a comparison with oleic acid.

The effects of arachidonic (AA) and oleic acids (OA) on proliferation, cytokine production and pleiotropic genes expression in Jurkat T cells were investigated. The following parameters were evaluated: cytotoxicity assessed by loss of membrane integrity and DNA fragmentation, cell proliferation as measured by [14C]-thymidine incorporation, production of IL-2, IL-4, IL-10, and INF-gamma, and expression of pleiotropic genes as determined by macroarray technique (83 genes in total). AA was more toxic for Jurkat cells than OA. However, the inhibiting effect of OA on Jurkat cells proliferation was more pronounced than that of AA. The reduction in the production of IL-2 and INF-gamma was more intense by OA (50 microM) than by AA (5 microM). The percentage of genes changed by the fatty acids was: 20.5% (17 genes) for AA (5 microM) and only 2.4% (2 genes) for OA (50 microM). AA markedly affected the expression of genes clustered as: signal transduction pathways, transcription factors and related genes, cell cycle, defense and repair, apoptosis, DNA synthesis, cell adhesion, cytoskeleton and related genes. In particular, AA induced marked changes in cell cycle, signal transduction, and anti-apoptosis genes expression. Therefore, the effect of AA on T-lymphocyte function does involve regulation of expression of important genes, whereas oleic acid did not markedly affect gene expression of Jurkat cells.

Arachidonic Acid↗

PubMed2003

Differential effects of short-chain fatty acids on proliferation and production of pro- and anti-inflammatory cytokines by cultured lymphocytes.

Short-chain fatty acids (SCFA) are produced by fermentation of water-soluble fiber by anaerobic bacteria in the large bowel. Fiber-rich diets decrease the risk of developing inflammatory bowel disease (IBD) and butyrate enemas are effective as a therapy in some patients. Crohn's disease, one form of IBD, appears to involve an exagerated T helper-1 (Th1) lymphocyte phenotype, characterised by production of interleukin (IL)-2 and interferon (IFN)-gamma, that drives the inflammation. To examine whether SCFA influence pro- and anti-inflammatory cytokine production, rat mesenteric lymph node lymphocytes were cultured in the presence of acetate (10 mM), butyrate (1.5 mM) or propionate (2 mM) and the production of cytokines in response to concanavalin A determined. Butyrate, but not acetate or propionate, inhibited lymphocyte proliferation and IL-2 production. Acetate and propionate were able to partly prevent the inhibitory effect of butyrate on IL-2 production. Acetate and propionate increased IFN-gamma production, whereas butyrate inhibited it. Acetate and propionate in combination were able to prevent the inhibitory effect of butyrate on IFN-gamma production. IL-4 was not detected in any cultures. Acetate and propionate increased IL-10 production, which was not affected by butyrate. It is concluded that butyrate significantly inhibits Th1-type responses and that this might explain the therapeutic effect of butyrate in IBD patients. Acetate and propionate have less marked modulatory actions, and in some cases have effects that oppose those of butyrate. A combination of the three SCFA causes a shift in the T helper lymphocyte phenotype towards a more anti-inflammatory phenotype and this might explain the protective effects of fiber.

Animals↗

PubMed2003

Palmitate modulates the early steps of insulin signalling pathway in pancreatic islets.

Insulin stimulates its own secretion and synthesis by pancreatic beta-cells. Although the exact molecular mechanism involved is unknown, changes in beta-cell insulin signalling have been recognized as a potential link between insulin resistance and its impaired release, as observed in non-insulin-dependent diabetes. However, insulin resistance is also associated with elevated plasma levels of free fatty acids (FFA) that are well known modulators of insulin secretion by pancreatic islets. This information led us to investigate the effect of FFA on insulin receptor signalling in pancreatic islets. Exposure of pancreatic islets to palmitate caused up-regulation of several insulin-induced activities including tyrosine phosphorylation of insulin receptor and pp185. This is the first evidence that short exposure of these cells to 100 microM palmitate activates the early steps of insulin receptor signalling. 2-Bromopalmitate, a carnitine palmitoyl-CoA transferase-1 inhibitor, did not affect the effect of the fatty acid. Cerulenin, an acylation inhibitor, abolished the palmitate effect on protein levels and phosphorylation of insulin receptor. This result supports the proposition that protein acylation may be an important mechanism by which palmitate exerts its modulating effect on the intracellular insulin signalling pathway in rat pancreatic islets.

Animals↗

PubMed2003

Palmitate acutely raises glycogen synthesis in rat soleus muscle by a mechanism that requires its metabolization (Randle cycle).

The acute effect of palmitate on glucose metabolism in rat skeletal muscle was examined. Soleus muscles from Wistar male rats were incubated in Krebs-Ringer bicarbonate buffer, for 1 h, in the absence or presence of 10 mU/ml insulin and 0, 50 or 100 microM palmitate. Palmitate increased the insulin-stimulated [(14)C]glycogen synthesis, decreased lactate production, and did not alter D-[U-(14)C]glucose decarboxylation and 2-deoxy-D-[2,6-(3)H]glucose uptake. This fatty acid decreased the conversion of pyruvate to lactate and [1-(14)C]pyruvate decarboxylation and increased (14)CO(2) produced from [2-(14)C]pyruvate. Palmitate reduced insulin-stimulated phosphorylation of insulin receptor substrate-1/2, Akt, and p44/42 mitogen-activated protein kinases. Bromopalmitate, a non-metabolizable analogue of palmitate, reduced [(14)C]glycogen synthesis. A strong correlation was found between [U-(14)C]palmitate decarboxylation and [(14)C]glycogen synthesis (r=0.99). Also, palmitate increased intracellular content of glucose 6-phosphate in the presence of insulin. These results led us to postulate that palmitate acutely potentiates insulin-stimulated glycogen synthesis by a mechanism that requires its metabolization (Randle cycle). The inhibitory effect of palmitate on insulin-stimulated protein phosphorylation might play an important role for the development of insulin resistance in conditions of chronic exposure to high levels of fatty acids.

Animals↗

PubMed2003

Glutamine delays spontaneous apoptosis in neutrophils.

Nuclear, mitochondrial, and plasma membrane events associated with apoptosis were investigated in rat neutrophils cultivated for 3, 24, and 48 h in the absence or presence of glutamine (0.5, 1.0, and 2.0 mM). Condensation of chromatin was reduced after 24 or 48 h of culture in the presence of glutamine compared with its absence as assessed by Hoechst 33342 staining. The level of Escherichia coli phagocytosis in the presence of glutamine was markedly increased compared with the level achieved by cells cultured in the absence of glutamine. Annexin V binding to externalized phosphatidylserine was reduced in the presence of glutamine. Sensitive fluorochrome rhodamine 123, as determined by fluorescence-activated cell sorting and confocal microscopy, was used to monitor loss of the mitochondrial transmembrane potential. In the absence of glutamine, neutrophils exhibited a marked reduction in the uptake of rhodamine 123. In the presence of 1.0 or 2.0 mM glutamine, the uptake of rhodamine was 20 or 38% higher, respectively. Similar effect was found in human neutrophils by measuring DNA fragmentation and mitochondrial transmembrane potential. Therefore, glutamine protects from events associated with triggering and executing apoptosis in both rat and human neutrophils.

Animals↗

PubMed2003

Effect of dexamethasone on neutrophil metabolism.

The effect of dexamethasone on glucose and glutamine metabolism was investigated. The consumption and oxidation of glucose and glutamine, and the production of glutamate and lactate were determined in neutrophils cultured for 3 h in the presence of dexamethasone. The activities and expression of glucose-6-phosphate dehydrogenase (G6PDH) and phosphate-dependent glutaminase were also determined under the same conditions. Addition of dexamethasone to the culture medium caused a significant increase of glucose consumption at 0.5 microm (123.9%) and 1.0 microm (78.3%) concentrations. In spite of this, however, glucose oxidation remained unchanged. The glucocorticoid did not change glutamine consumption but caused a significant increase of glutamate production and did not alter glutamine oxidation. Dexamethasone-treated neutrophils had a significant decrease of G6PDH activity and expression in particular at 1.0 microm concentration. Phosphate- dependent glutaminase activity was also decreased (about 34%) by dexamethasone treatment. A similar effect was observed on glutaminase expression as indicated by RT-PCR analysis. Thus, the effect of dexamethasone on neutrophil metabolism was particularly noticeable with respect to G6PDH and glutaminase activities where a decrease in the respective mRNA levels was demonstrated.

Animals↗

PubMed2003

Glutamine and glutamate--their central role in cell metabolism and function.

Glucose is widely accepted as the primary nutrient for maintenance and promotion of cell function. However, we propose that the 5-carbon amino acids, glutamine and glutamate, should be considered to be equally important for maintenance and promotion of cell function. The functions of glutamine are many and include: substrate for protein synthesis, anabolic precursor for muscle growth, acid-base balance in the kidney, substrate for ureogenesis in the liver, substrate for hepatic and renal gluconeogenesis, an oxidative fuel for intestine and cells of the immune system, inter-organ nitrogen transport, precursor for neurotransmitter synthesis, precursor for nucleotide and nucleic acid synthesis and precursor for glutathione production. Many of these functions are connected to the formation of glutamate from glutamine. We propose that the unique properties regarding concentration and routes of metabolism of these amino acids allow them to be used for a diverse array of processes related to the specialized function of each of the glutamine utilizing cells. In this review we highlight the specialized aspects of glutamine/glutamate metabolism of different glutamine-utilizing cells and in each case relate key aspects of metabolism to cell function.

Animals↗

PubMed2003

Arachidonic acid cytotoxicity: can arachidonic acid be a physiological mediator of cell death?

Arachidonic acid is a polyunsaturated fatty acid that mediates inflammation and the functioning of several organs and systems either directly or upon its conversion into eicosanoids. However, arachidonic acid is found to be cytotoxic in vitro at concentrations that overlap physiological ones. It is tempting therefore to speculate that arachidonic acid may be a physiological inducer of apoptosis and that such cytotoxic action may be another of its roles in vivo. Nevertheless its pro-inflammatory and oxidative stress-inducing features are characteristic of necrosis and pathological conditions. We hereby review the cytotoxic action of arachidonic acid, indicate the possible pathways that lead to cell death and contemplate the cytotoxic role of arachidonic acid in vivo.

Animals↗

PubMed2003

Human cutaneous melanoma expresses a significant phosphate-dependent glutaminase activity: a comparison with the surrounding skin of the same patient.

The protein content and the activity and type of phosphate-dependent glutaminase were determined in freshly pigmented lesions obtained from human melanoma and adjacent skin. Significant phosphate-dependent glutaminase activity was found in both the melanoma and non-pigmented adjacent skin areas. A comparison between the pigmented and adjacent skin areas suggests the occurrence of gradual metabolic changes that result in an increased protein content in the centre of the neoplasia. The presence of a kidney-type glutaminase (K(m) of 2-5 mm) indicates a high sensitivity of the melanoma to variations in glutamine plasma levels (0.6 to 1 mm). These data lead us to postulate that glutamine supply is an important factor for melanoma cell proliferation, being a source of nitrogen for DNA and RNA synthesis. The intense neovascularization observed in melanoma ensures the oxygen supply that is required for glutamine oxidation. These findings support the proposition that glutamine is an important fuel for melanoma.

Glutaminase↗