Proteomic analysis of an orthotopic neuroblastoma xenograft animal model

Neuroblastoma is the most common extracranial solid tumour of childhood and comprises up to 50% of malignancies among infants. There is a great need of designing novel therapeutic strategies and proteome analysis is one approach for defining markers useful for tumour diagnosis, as well as molecular targets for novel experimental therapies. We started by comparing healthy adrenal glands (which are the election organs developing primary neuroblastoma, NB, tumours) and adrenal glands carrying primary NB tumours, taken from nude mice. Standard maps of healthy and tumour samples were generated by analysis with the PDQuest software. The comparison between such maps showed up- and down-regulation of 84 polypeptide chains, out of a total of 700 spots detected by a fluorescent stain, Sypro Ruby. Spots that were differentially expressed between the two groups, were analysed by MALDI-TOF mass spectrometry and 14 of these spots were identified so far. Among these proteins, of particular interest are the down-regulated proteins adrenodoxin (21-folds), carbonic anhydrase III (eight-folds) and aldose reductase related protein I (eight-folds), as well as the up-regulated protein peptidyl-propyl cis-trans isomerase A (five-folds). Moreover new proteins, which were absent in control samples, were expressed in tumour samples, such as nucleophosmin (NPM) and stathmin (oncoprotein 18).     

Hepcidin assay in serum by SELDI-TOF-MS and other approaches

Hepcidin, a liver peptide hormone, is the central regulator of iron homeostasis. Hepcidin synthesis is modulated by iron stores, so that iron repletion increases its levels to prevent pathological overload, while iron deficiency strongly inhibits hepcidin to allow an increase in iron absorption from duodenal cells. The emerging pivotal role of hepcidin in iron homeostasis, along with its important links with basic pathways like inflammation, makes the availability of an accurate hepcidin assay as a potentially powerful investigative tool to improve our understanding as well as our diagnostic/prognostic capabilities in many human diseases. There has been a great interest worldwide in developing a reliable and widely applicable assay of the hormone in biological fluids. Being optimal for low-molecular-weight biomarkers, SELDI-TOF-MS has emerged as a valid tool for hepcidin assay. Here we review recent results obtained with this technique, as well as with other Mass Spectrometry-based and immunological methods.     

Two-dimensional mapping as a tool for classification of green coffee bean species

Two species of the genus Coffea, Coffea arabica (Colombia) and Coffea canephora (Indiano Robusta) were analysed by two-dimensional (2-D) maps in order to obtain fingerprints of the expressed polypeptide chains and to determine which ones would characterize the two species. Green beans were milled under liquid nitrogen. A dry powder was produced by three different extraction protocols aimed at eliminating interfering substances (polyphenols). A reduced powder was produced by two successive extractions performed in acetone. Trichloroacetic acid (TCA; 10% w/v) and beta-mercaptoethanol (0.07% v/v) in acetone were used for the first extraction (a) and 10% w/v TCA in acetone was used for the second extraction (b). Proteins were then solubilized in a solution (40 microL per 1 mg powder) containing 7 M urea, 2 M thiourea, 3% w/v 3-(3-cholamidopropyldimethyl-amino)-1-propanesulfate, 1% v/v carrier ampholytes, 40 mM Tris, 5 mM tributylphosphine and 10 mM acrylamide as alkylating agent. Following incubation at room temperature for 1 hour and centrifugation (7000 rpm for 20 minutes), the supernatant was used for 2-D electrophoresis. The proteins were revealed by Sypro Ruby staining. Master maps of the five replicas of each species were compared by PDQuest analysis. The results of this differential proteome analysis were: sixteen proteins were expressed solely in C. canephora (var. Indiano Robusta) and five proteins were only found in C. arabica (var. Colombia). Another eight proteins were up-regulated in C. canephora (var. Indiano Robusta) in comparison to C. arabica (var. Colombia) and one was down-regulated in the same comparison. A number of these polypeptide chains were further characterized by mass spectrometry in the matrix-assisted laser desorption/ionisation-time of flight mode. Additionally, considering the low number of protein sequences of Coffea present in the databases we also investigated some spots with a more powerful tool, reversed phase-high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry, thus obtaining an internal peptide sequence. The general properties of the identified proteins are presented and discussed.     

Increased Serum Hepcidin Levels in Subjects with the Metabolic Syndrome: A Population Study

The recent discovery of hepcidin, the key iron regulatory hormone, has changed our view of iron metabolism, which in turn is long known to be linked with insulin resistant states, including type 2 diabetes mellitus and the Metabolic Syndrome (MetS). Serum ferritin levels are often elevated in MetS (Dysmetabolic hyperferritinemia - DHF), and are sometimes associated with a true mild-to-moderate hepatic iron overload (dysmetabolic iron overload syndrome - DIOS). However, the pathophysiological link between iron and MetS remains unclear. This study was aimed to investigate, for the first time, the relationship between MetS and hepcidin at population level. We measured serum hepcidin levels by Mass Spectrometry in 1,391 subjects from the Val Borbera population, and evaluated their relationship with classical MetS features. Hepcidin levels increased significantly and linearly with increasing number of MetS features, paralleling the trend of serum ferritin. In multivariate models adjusted for relevant variables including age, C-Reactive Protein, and the HFE C282Y mutation, ferritin was the only significant independent predictor of hepcidin in males, while in females MetS was also independently associated with hepcidin. Overall, these data indicate that the fundamental iron regulatory feedback is preserved in MetS, i.e. that hepcidin tends to progressively increase in response to the increase of iron stores. Due to recently discovered pleiotropic effects of hepcidin, this may worsen insulin resistance and contribute to the cardiovascular complications of MetS.     

Proteomic analysis of S-nitrosylated proteins in Arabidopsis thaliana undergoing hypersensitive response

Nitric oxide (NO) has a fundamental role in the plant hypersensitive disease resistance response (HR), and S-nitrosylation is emerging as an important mechanism for the transduction of its bioactivity. A key step toward elucidating the mechanisms by which NO functions during the HR is the identification of the proteins that are subjected to this PTM. By using a proteomic approach involving 2-DE and MS we characterized, for the first time, changes in S-nitrosylated proteins in Arabidopsis thaliana undergoing HR. The 16 S-nitrosylated proteins identified are mostly enzymes serving intermediary metabolism, signaling and antioxidant defense. The study of the effects of S-nitrosylation on the activity of the identified proteins and its role during the execution of the disease resistance response will help to understand S-nitrosylation function and significance in plants.     

Urinary hepcidin identifies a serum ferritin cut-off for iron supplementation in young athletes: a pilot study

The use of iron supplements should be a judicious choice, primarily when considering the possible risks deriving from an unjustified treatment. In trained athletes, levels of ferritin between 15 and 30 microg/L are frequently observed. Within this ferritin range, the usefulness of iron supplementation is still controversial. The aim of the present study is to evaluate the clinical usefulness of hepcidin assessment in the analysis of the iron status of young non-anemic athletes. Fifty young athletes were enrolled. The subjects were divided into 4 groups according to their ferritin levels. No statistically significant difference was found regarding hepcidin levels between athletes with ferritin lower than 15 microg/L and those in the 15-30 microg/L range. Similarly, no difference was found between athletes with ferritin higher than 50 microg/L and those in the 30-50 microg/L range. On the contrary, statistically significant differences were found between athletes with ferritin levels ranging from 15 to 30 microg/L and those in the 30-50 microg/L range. The present study suggests that serum ferritin levels below 30 microg/L indicate an asymptomatic iron deficiency status inhibiting hepcidin expression and that 30 microg/L should be considered the ferritin cut-off when considering an iron supplementation in young athletes.     

Over the hills and far away: New plant records for the Guayana Shield in Brazil

The Guayana Shield is one of the oldest geological formations in South America, ranging from southern Colombia, Venezuela, and the Guianas to the extreme north of the Brazilian states of Amazonas and Roraima. Because of its ancient origin and isolation from other mountain ranges in South America, it harbors a rich flora with high levels of endemism. Recent expeditions to remote areas in the Brazilian portion of the Guayana Shield generated many new herbarium collections. Through these efforts, we report the first records for Brazil of 57 species and infraspecific taxa of vascular plants. In most cases, the taxa are narrowly distributed and were previously known from nearby populations in other countries. In other cases, however, the new Brazilian records expand considerably the known geographical distributions of the taxa.     

Biochemical and ecophysiological responses to manganese stress by ectomycorrhizal fungus <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis> and in association with <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

At relatively low concentrations, the element manganese (Mn) is essential for plant metabolism, especially for photosynthesis and as an enzyme antioxidant cofactor. However, industrial and agricultural activities have greatly increased Mn concentrations, and thereby contamination, in soils. We tested whether and how growth of Pisolithus tinctorius is influenced by Mn and glucose and compare the activities of oxidative stress enzymes as biochemical markers of Mn stress. We also compared nutrient accumulation, ecophysiology, and biochemical responses in Eucalyptus grandis which had been colonized by the ectomycorrhizal Pisolithus tinctorius with those which had not, when both were exposed to increasing Mn concentrations. In vitro experiments comprised six concentrations of Mn in three concentrations of glucose. In vivo experiments used plants colonized by Pisolithus tinctorius, or not colonized, grown with three concentrations of Mn (0, 200, and 1000 μM). We found that fungal growth and glucose concentration were correlated, but these were not influenced by Mn levels in the medium. The anti-oxidative enzymes catalase and glutathione S-transferase were both activated when the fungus was exposed to Mn. Also, mycorrhizal plants grew more and faster than non-mycorrhizal plants, whatever Mn exposure. Photosynthesis rate, intrinsic water use efficiency, and carboxylation efficiency were all inversely correlated with Mn concentration. Thus, we originally show that the ectomycorrhizal fungus provides protection for its host plants against varying and potentially toxic concentrations of Mn.     

Genetic Variation in Three Endangered Species of Encholirium (Bromeliaceae) from Cadeia do Espinhaço, Brazil, Setected using RAPD Markers

Encholirium is a Brazilian genus of Bromeliaceae that occurs exclusively in rocky landscapes. This work aimed to generate basic information for the conservation of three Encholirium species that are endemic to the rocky mountains of Cadeia do Espinhaço, employing population genetic analyses. E. pedicellatum and E. biflorum have only one very small population each, both occurring in unprotected, private land sites, being critically endangered. E. subsecundum is more widespread, and some of its populations dwell in protected areas. Five Random Amplified Polymorphic DNA (RAPD) primers generated approximately 60 polymorphic bands for each species. This technique demonstrated the presence of a single RAPD profile for every individual sampled (except for one clone found in E. biflorum). High levels of genetic variability were not expected, due to the clonal habit of the plants and small size of the populations. Populations of E. biflorum and E. pedicellatum presented, respectively, 16.06% (Φ _st  = 0.16, p<0.001) and 8.44% (Φ _st  = 0.08, p<0.001) of the total genetic diversity attributable to genetic differences among groups within the populations. In E. subsecundum, 14.52% (Φ _st  = 0.15, p<0.001) of the total genetic diversity was found among populations. Estimates of the Shannon’s Diversity Index provided similar results. These results are valuable for the development of conservation strategies.