The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins

Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.     

Recent coral facies of the Indian Ocean Coast of Somalia with an interim check list of corals

Summary From a study of two areas, Jesira and the Bajuni Archipelago, about 400 km apart, a general pattern can be established for the Recent facies, together with the morphological and taxonomic features of the corals. Present day coral development is characterized by true fringing reefs in the Bajuni Archipelago and by scattered patches and knolls in the Jesira area. The coral fauna, consisting of 27 genera and 63 species so far (including all uncertainties, but not sight records), is rather poor, though coral communities are locally well developed. These figures probably reflect incomplete study and sampling. Although comparison with other areas may therefore be premature, a preliminary biogeographical analysis suggests that this fauna is more closely related to that of the Red Sea than to East Africa and the Seychelles. This differs from other published biogeographical work on Indian Ocean coral faunas, but further study of the corals in this and neighbouring areas of the Indian Ocean is needed in order to resolve this apparent anomaly.     

Phylogenetic analysis and a time tree for a large drosophilid data set (Diptera: Drosophilidae)

Drosophila is the genus responsible for the birth of experimental genetics, but the taxonomy of drosophilids is difficult because of the overwhelming diversity of the group. In this study, we assembled sequences for 358 species (14 genera, eight subgenera, 57 species groups, and 65 subgroups) to generate a maximum‐likelihood topology and a Bayesian timescale. In addition to sampling an unprecedented diversity of Drosophila lineages, our analyses incorporated a geographical perspective because of the high levels of endemism. In our topology, Drosophila funebris (Fabricius, 1787) (the type species of Drosophila) is tightly clustered with the pinicola subgroup in a North American clade within subgenus Drosophila. The type species of other drosophilid genera fall within the Drosophila radiation, presenting interesting prospects for the phylogenetic taxonomy of the group. Our timescale suggests that a few drosophilid lineages survived the Cretaceous–Palaeogene (K‐Pg) extinction. The drosophilid diversification began during the Palaeocene in Eurasia, but peaked during the Miocene, an epoch of drastic climatic changes. The most recent common ancestor of the clades corresponding to subgenera Sophophora and Drosophila lived approximately 56 Mya. Additionally, Hawaiian drosophilids diverged from an East Asian lineage approximately 26 Mya, which is similar to the age of the oldest emerging atoll in the Hawaiian–Emperor Chain. Interestingly, the time estimates for major geographical splits (New World versus Asia and Africa versus Asia) were highly similar for independent lineages. These results suggest that vicariance played a significant role in the radiation of fruit flies. © 2013 The Linnean Society of London     

Mapping of p140Cap Phosphorylation Sites: The EPLYA and EGLYA Motifs Have a Key Role in Tyrosine Phosphorylation and Csk Binding,and Are Substrates of the Abl Kinase

Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.     

The Effects of Aging on Conflict Detection

Several cognitive changes characterize normal aging; one change regards inhibitory processing and includes both conflict monitoring and response suppression. We attempted to segregate these two aspects within a Go/No-go task, investigating three age categories. Accuracy, response times and event-related potentials (ERPs) were recorded. The ERP data were analyzed, and the Go and No-go trials were separated; in addition, the trials were organized in repeat trials (in which the subjects repeated the action delivered in the previous trial) and switch trials (in which the subjects produced a response opposite to the previous response). We assumed that the switch trials conveyed more conflict than the repeat trials. In general, the behavioral data and slower P3 latencies confirmed the well-known age-related speed/accuracy trade-off. The novel analyses of the repeat vs. switch trials indicated that the age-related P3 slowing was significant only for the high conflict condition; the switch-P3 amplitude increased only in the two older groups. The ‘aging switch effect’ on the P3 component suggests a failure in the conflict conditions and likely contributes to a generalized dysfunction. The absence of either a switch effect in the young group and the P3 slowing in middle-aged group indicate that switching was not particularly demanding for these participants. The N2 component was less sensitive to the repeat/switch manipulation; however, the subtractive waves also enhanced the age effects in this earlier time window. The topographic maps showed other notable age effects: the frontal No-go N2 was nearly undetectable in the elderly; in the identical time window, a large activity in the posterior and prefrontal scalp regions was observed. Moreover, the prefrontal activity showed a negative correlation with false alarms. These results suggest that the frontal involvement during action suppression becomes progressively dysfunctional with aging, and additional activity was required to reach a good level of accuracy.     

Palmitoylethanolamide Treatment Reduces Blood Pressure in Spontaneously Hypertensive Rats: Involvement of Cytochrome P450-Derived Eicosanoids and Renin Angiotensin System

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α agonist, has been demonstrated to reduce blood pressure and kidney damage secondary to hypertension in spontaneously hypertensive rat (SHR). Currently, no information is available concerning the putative effect of PEA on modulating vascular tone. Here, we investigate the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids, CYP-dependent arachidonic acid metabolites, as endothelium-derived hyperpolarizing factors (EDHF), and renin angiotensin system (RAS) modulation. To achieve this aim SHR and Wistar-Kyoto rats were treated with PEA (30 mg/kg/day) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF-mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure CYP2C23 and CYP2J2, the key isoenzymes in the formation of epoxyeicosatrienoic acids, and the soluble epoxide hydrolase, which is responsible for their degradation in the corresponding diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme and angiotensin receptor 1 expression. Here, we showed that EDHF-mediated dilation in response to acetylcholine was increased in mesenteric beds of PEA-treated SHR. Western blot analysis revealed that the increase in CYP2C23 and CYP2J2 observed in SHR was significantly attenuated in mesenteric beds of PEA-treated SHR, but unchanged in the carotids. Interestingly, in both vascular tissues, PEA significantly decreased the soluble epoxide hydrolase protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid, implying a reduction in epoxyeicosatrienoic acid hydrolisis. Moreover, PEA treatment down-regulated angiotensin receptor 1 and angiotensin converting enzyme expression, indicating a reduction in angiotensin II-mediated effects. Consistently, a damping of the activation of angiotensin receptor 1 underlying pathways in mesenteric beds was shown in basal conditions in PEA-treated SHR. In conclusion, our data demonstrate the involvement of epoxyeicosatrienoic acids and renin angiotensin system in the blood pressure lowering effect of PEA.     

Effects of 6-month daily supplementation with oral beta-carotene in combination or not with benzo[a]pyrene on cell-cycle markers in the lung of ferrets

Epidemiological studies have demonstrated that people who eat more fruits and vegetables (rich in carotenoids) and people who have higher serum beta-carotene (BC) levels have a lower risk of cancer, particularly lung cancer. However, the two main human intervention studies of BC supplementation (the ATBC and the CARET trials) revealed an increased risk of lung cancer among smokers and asbestos workers. Previous studies carried out in the ferret have reported that BC effects are related to dose. Here, we treated ferrets with two concentrations of oral BC (0.8 and 3.2 mg/kg body weight per day) for 6 months, using BC in a formulation also containing dl-alpha-tocopherol and ascorbyl palmitate. The effect of the smoke-derived carcinogenic agent benzo[a]pyrene (BP), with or without low-dose BC, was also analysed. We determined the protein levels and mRNA expression levels of activator protein 1 (c-Jun and c-Fos), c-Myc, cyclin D1, proliferating cellular nuclear antigen and retinoic acid receptor beta. We did not find higher levels of cell proliferation markers in the lung of ferrets treated with BC or signals of squamous metaplasia lesions either. On the other hand, although no evident signals of pulmonary carcinogenesis were observed in animals exposed to BP, BC supplementation in these animals may prevent against excess cell proliferation, since this reestablishes Jun protein and cyclin D1 mRNA levels in the lung of BP-exposed animals. In summary, these results show that the combination of BC with alpha-tocopherol and ascorbyl palmitate does not induce pro-oxidant effects in the lung of ferrets.     

Human milk oligosaccharides: an enzymatic protection step simplifies the synthesis of 3'- and 6'-O-sialyllactose and their analogues

We describe a chemo-enzymatic synthesis of 3'- and 6'-O-sialyllactose, two trisaccharides occurring in the 'acidic fraction' of the human milk oligosaccharides and endowed with potential antiadhesive activity. The key step is the highly regioselective 6'-O-acylation of benzyllactoside, which gave access to suitably protected lactose building blocks to be used as acceptors in the sialylation reaction. Moreover, the synthesis of the carboxymethyl and sulfo analogues of the title compounds is reported.