Proteomic and ionomic profiling reveals significant alterations of protein expression and calcium homeostasis in cystic fibrosis cells

Cystic fibrosis (CF) is an autosomal recessive disorder associated with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and defective chloride transport across the epithelial cell membranes. Abnormal epithelial ion transport is the primary cause of persistent airway infections and chronic inflammation in CF patients. In order to gain further insight into the mechanisms of epithelial dysfunctions linked to CFTR mutations, we performed and integrated proteomic and ionomic analysis of human bronchial epithelial IB3-1 cells and compared them with a CFTR-complemented isogenic cell line (C38). Aside from changes that were consistent with known effects related to CFTR mutations, such as differences in glycolytic and gluconeogenic pathways and unfolded protein responses, differential proteomics highlighted significant alteration of protein expression and, in particular, of the 14-3-3 signalling pathway that is known to be involved in cellular calcium (Ca) homeostasis. Of note, restoring chloride efflux by acting on Ca cellular homeostasis has been shown to be a promising therapeutic intervention for CF. Ionomic analysis showed significant changes in the IB3-1 element profile compared with C38 cells and in particular we observed an increase of intracellular Ca that significantly correlates with intracellular zinc (Zn) levels, suggesting a synergistic role of Ca and Zn influx. This finding is particularly intriguing because Zn has been reported to be effective in CF treatment increasing Ca influx. Taken together, our proteomic and ionomic data reveal that CFTR mutation sets in motion endogenous mechanisms counteracting impaired chloride transport mainly acting on epithelial ion transport and increasing intracellular Ca, suggesting potential links between protein expression and this response

N-acetylcysteine reduces oxidative stress in sickle cell patients

Oxidative stress is of importance in the pathophysiology of sickle cell disease (SCD). In this open label randomized pilot study the effects of oral N-acetylcysteine (NAC) on phosphatidylserine (PS) expression as marker of cellular oxidative damage (primary end point), and markers of hemolysis, coagulation and endothelial activation and NAC tolerability (secondary end points) were studied. Eleven consecutive patients (ten homozygous [HbSS] sickle cell patients, one HbSβ0-thalassemia patient) were randomly assigned to treatment with either 1,200 or 2,400 mg NAC daily during 6 weeks. The data indicate an increment in whole blood glutathione levels and a decrease in erythrocyte outer membrane phosphatidylserine exposure, plasma levels of advanced glycation end-products (AGEs) and cell-free hemoglobin after 6 weeks of NAC treatment in both dose groups. One patient did not tolerate the 2,400 mg dose and continued with the 1,200 mg dose. During the study period, none of the patients experienced painful crises or other significant SCD or NAC related complications. These data indicate that N-acetylcysteine treatment of sickle cell patients may reduce SCD related oxidative stress

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Selenium independent glutathione peroxidase activity associated with cationic forms of glutathione transferase in human heart

Glutathione peroxidase activity with both hydrogen peroxide and cumene hydroperoxide was measured in the cytosolic fractions prepared from five human hearts obtained from post-mortem victims. In all the samples the activity with cumene hydroperoxide was higher than that obtained with hydrogen peroxide, suggesting that the selenium-independent glutathione peroxidase could also be present in this tissue. To determine its presence in heart tissue we fractionated the cardiac cytosol fraction on a column of Sephadex G-100 and measured glutathione peroxidase activity with both the substrates. Glutathione transferase activity was measured with 1-chloro-2,4-dinitrobenzene in the fractionated cytosol. The results indicated that a selenium-independent glutathione peroxidase activity was present (about 30% of total activity). Fractionation of the cytosol by gel filtration showed that peroxidase activity co-eluted with glutathione transferase activity. Subsequently the fractions containing glutathione transferase and selenium-independent glutathione peroxidase activity obtained from gel filtration experiments were passed through an affinity column and analyzed by isoelectric focusing. It was found that the selenium-independent glutathione peroxidase copurified with three isoenzymes of glutathione transferase which had a pI of 9.2, 8.9 and 8.6 respectively. In contrast the acidic isoenzymes of glutathione transferase lacked peroxidase activity. It is suggested that the selenium-independent glutathione peroxidase may play an important role in neutralizing oxygen toxicity in heart when the selenium-dependent glutathione peroxidase activity is impaired

Selenium independent glutathione peroxidase activity associated with cationic forms of glutathione transferase in human heart

Glutathione peroxidase activity with both hydrogen peroxide and cumene hydroperoxide was measured in the cytosolic fractions prepared from five human hearts obtained from post-mortem victims. In all the samples the activity with cumene hydroperoxide was higher than that obtained with hydrogen peroxide, suggesting that the selenium-independent glutathione peroxidase could also be present in this tissue. To determine its presence in heart tissue we fractionated the cardiac cytosol fraction on a column of Sephadex G-100 and measured glutathione peroxidase activity with both the substrates. Glutathione transferase activity was measured with 1-chloro-2,4-dinitrobenzene in the fractionated cytosol. The results indicated that a selenium-independent glutathione peroxidase activity was present (about 30% of total activity). Fractionation of the cytosol by gel filtration showed that peroxidase activity co-eluted with glutathione transferase activity. Subsequently the fractions containing glutathione transferase and selenium-independent glutathione peroxidase activity obtained from gel filtration experiments were passed through an affinity column and analyzed by isoelectric focusing. It was found that the selenium-independent glutathione peroxidase copurified with three isoenzymes of glutathione transferase which had a pI of 9.2, 8.9 and 8.6 respectively. In contrast the acidic isoenzymes of glutathione transferase lacked peroxidase activity. It is suggested that the selenium-independent glutathione peroxidase may play an important role in neutralizing oxygen toxicity in heart when the selenium-dependent glutathione peroxidase activity is impaired

Proteomic analysis of cryoconserved bull sperm to enhance ERCR classification scores of fertility

Breeding of dairy cattle for high production and the reproductive management of herd is the biggest problem and it accounts for a large part on costs of production. A negative association has been observed between the level of livestock production and fertility. This is linked both to genetic factors (inbreeding and high production) and physiological factors (metabolic by high production)1. A lot of resources have been used for enhancement of cattle fertility but few studies and interventions are reported to control and to enhance the effect on the bull reproductive efficiency. As the patterns of selection and reproductive management of dairy cattle is based on the use of artificial insemination (AI) it is easy to understand the importance of assessing the level of fertility of bull breeder. One method of evaluating relative sire fertility currently used is the estimated relative conception rate (ERCR). ERCR is the difference in conception rate (nonreturn rate at 56 day) of a sire compared with other AI sires used in the same herd2. In this work the nonreturn rate was estimated at 56 d for first insemination of lactating cows (www.\u200banafi.\u200bit). At present, validation of genomic markers that are able to predict with high confidence high or low fertility of a given sire it is very difficult using population estimates of sire fertility. The reason is because these methods do not measure the bull \u2018true fertility\u20193. To unravel the biological display of the bull genome, proteomics, that focus at the protein level could lead to the development of novel biomarkers that may allow for detection of bull fertility levels4,5. The aim of this study is to evaluate, through the differential proteome analysis, changes in protein expression profiles of spermatozoa from bulls with high fertility (high ERCR score) and low fertility (low ERCR score) in order to identify possible protein markers to be used as indices of fertilit

Gastrointestinal tolerability of ibuprofen administered in two pharmaceutical formulations

Ibuprofen (CAS 15687-27-1) is a nonsteroidal antiinflammatory drug endowed with analgesic, antiinflammatory and antipyretic activity. The main side effect of ibuprofen and nonsteroidal antiinflammatory agents is addressed to disturbances of the gastrointestinal tract, like gastric pyrosis, gastric and intestinal damage. A pharmaceutical formulation of ibuprofen in fast melting tablets consisting in gastroprotected microgranules to be ingested without concomitant water intake (Cibalginadue Fast, hereinafter referred to as test) was compared in this trial with a formulation of ibuprofen in tablets (reference) on 18 healthy volunteers in terms of gastrointestinal and general tolerability. Both the formulations are present on the market. The two formulations were administered according to a two-period, two-formulation, two-sequence, cross-over design in repeated dose regimen to steady state with wash-out. The dose was 400 mg (2 strengths) b.i.d. over 7 days. Before, during and after each study period general tolerability was carefully checked from blood/urine biochemical parameters, adverse events experienced and vital signs. The target parameters were the gastric permeability to sucrose and the intestinal permeability to lactulose and mannitol, which were administered orally and assayed in the urine excreted during a 6-h period. Urinary excretion > 0.15% of sucrose and > 0.04 of the lactulose to mannitol ratio are considered expression of increased gastric and intestinal permeability, respectively. Three volunteers treated with the reference showed an increased gastric permeability > 0.15%. Neither other gastric nor intestinal increased permeability was detected. Occult blood in faeces was negative in all the cases. The incidence of adverse effects experienced was higher with the reference (9 volunteers) than with the test (5 volunteers). In details gastric pyrosis was experienced by six volunteers treated with the reference and only by two volunteers treated with the test. The whole tolerability was better with the test formulation than with the reference, even if these differences did not reach any statistically significant degree. The better tolerability of the test was attributed to its gastroprotection

Glutathione S-transferase, similar to sigma class, from skin secretion of Xenopus laevis

IF 1.79