An automated method for scanning LC-MS data sets for significant peptides and proteins, including quantitative profiling and interactive confirmation

Differential quantification of proteins and peptides by LC-MS is a promising method to acquire knowledge about biological processes, and for finding drug targets and biomarkers. However, differential protein analysis using LC-MS has been held back by the lack of suitable software tools. Large amounts of experimental data are easily generated in protein and peptide profiling experiments, but data analysis is time-consuming and labor-intensive. Here, we present a fully automated method for scanning LC-MS/MS data for biologically significant peptides and proteins, including support for interactive confirmation and further profiling. By studying peptide mixtures of known composition, we demonstrate that peptides present in different amounts in different groups of samples can be automatically screened for using statistical tests. A linear response can be obtained over almost 3 orders of magnitude, facilitating further profiling of peptides and proteins of interest. Furthermore, we apply the method to study the changes of endogenous peptide levels in mouse brain striatum after administration of reserpine, a classical model drug for inducing Parkinson disease symptoms.     

An investigation of the peroxidase activity of <Emphasis Type="Italic">Vitreoscilla</Emphasis> hemoglobin

In order to investigate the ability of the Vitreoscilla hemoglobin (VHb) to act as a peroxidase, the protein was overexpressed in Escerichia coli and purified using a 6xHis-tag. The peroxidase activity of VHb was studied using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferrocene carboxylic acid (FcCOOH) dopamine and l-dopa as substrates. The effects of external agents such as pH, salt concentration/ionic strength, and the thermal stability of VHb on the catalytic activity were assessed. The optimum pH for VHb using ABTS as a substrate was estimated to be 6–7. The VHb protein proved to be stable up to 80 °C, as judged by its peroxidase activity. Furthermore, NaCl concentrations up to 100 mM did not exert any significant effect on the activity. The catalytic activity against ABTS and FcCOOH was similar to that measured for horseradish peroxidase, whereas in the case of the phenolic substrates dopamine and l-dopa the activity was several orders of magnitude lower. The Michaelis constants, were in good agreement with the data for human and bovine hemoglobin. No activity could be detected for the negative controls lacking VHb. These results demonstrate that VHb exhibits peroxidase activity, a finding in line with the hypothesis that VHb has cellular functions beyond the role as an oxygen carrier. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Malin Kvist and Ekaterina S. Ryabova contributed equally to this work.     

Ngn2 and Nurr1 act in synergy to induce midbrain dopaminergic neurons from expanded neural stem and progenitor cells

Parkinson's Disease (PD) is a debilitating motor function disorder due primarily to a loss of midbrain dopaminergic neurons and a subsequent reduction in dopaminergic innervation of the striatum. Several attempts have been made to generate dopaminergic neurons from progenitor cell populations in vitro for potential use in cell replacement therapy for PD. However, expanding cells from fetal brain with retained potential for dopaminergic differentiation has proven to be difficult. In this study, we sought to generate mesencephalic dopaminergic (mesDA) neurons from an expanded population of fetal mouse ventral midbrain (VM) progenitors through the use of retroviral gene delivery. We over-expressed Ngn2 and Nurr1, two genes present in the ventral midbrain and important for normal development of mesDA neurons, in multi-passaged neurosphere-expanded midbrain progenitors. We show that over-expression of Ngn2 in these progenitors results in increased neuronal differentiation but does not promote mesDA formation. We also show that over-expression of Nurr1 alone is sufficient to generate tyrosine hydroxylase (TH) expressing cells with an immature morphology, however the cells do not express any additional markers of mesDA neurons. Over-expression of Nurr1 and Ngn2 in combination generates morphologically mature TH-expressing neurons that also express additional mesencephalic markers.     

Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus influenzae lipopolysaccharide

We report the novel pattern of lipopolysaccharide (LPS) expressed by two disease-associated nontypeable Haemophilus influenzae strains, 1268 and 1200. The strains express the common structural motifs of H. influenzae; globotetraose [beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and its truncated versions globoside [alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and lactose [beta-d-Galp-(1-->4)-beta-d-Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an alpha-d-Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS molecule. Previously these motifs had been found linked only to either the proximal heptose (HepI) or HepIII of the triheptosyl inner-core moiety l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. This novel finding was obtained by structural studies of LPS using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material, as well as electrospray ionization-multiple-step tandem mass spectrometry on permethylated dephosphorylated oligosaccharide material. A lpsA mutant of strain 1268 expressed LPS of reduced complexity that facilitated unambiguous structural determination. Using capillary electrophoresis-ESI-MS/MS we identified sialylated glycoforms that included sialyllactose as an extension from HepII, this is a further novel finding for H. influenzae LPS. In addition, each LPS was found to carry phosphocholine and O-linked glycine. Nontypeable H. influenzae strain 1200 expressed identical LPS structures to 1268 with the difference that strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS has glycine at the same position.     

Interaction Effects of Ambient UV Radiation and Nutrient Limitation on the Toxic Cyanobacterium <Emphasis Type="Italic">Nodularia spumigena</Emphasis>

Nodularia spumigena is one of the dominating species during the extensive cyanobacterial blooms in the Baltic Sea. The blooms coincide with strong light, stable stratification, low ratios of dissolved inorganic nitrogen, and dissolved inorganic phosphorus. The ability of nitrogen fixation, a high tolerance to phosphorus starvation, and different photo-protective strategies (production of mycosporine-like amino acids, MAAs) may give N. spumigena a competitive advantage over other phytoplankton during the blooms. To elucidate the interactive effects of ambient UV radiation and nutrient limitation on the performance of N. spumigena, an outdoor experiment was designed. Two radiation treatments photosynthetic active radiation (PAR) and PAR +UV-A + UV-B (PAB) and three nutrient treatments were established: nutrient replete (NP), nitrogen limited (−N), and phosphorus limited (−P). Variables measured were specific growth rate, heterocyst frequency, cell volume, cell concentrations of MAAs, photosynthetic pigments, particulate carbon (POC), particulate nitrogen (PON), and particulate phosphorus (POP). Ratios of particulate organic matter were calculated: POC/PON, POC/POP, and PON/POP. There was no interactive effect between radiation and nutrient limitation on the specific growth rate of N. spumigena, but there was an overall effect of phosphorus limitation on the variables measured. Interaction effects were observed for some variables; cell size (larger cells in −P PAB compared to other treatments) and the carotenoid canthaxanthin (highest concentration in −N PAR). In addition, significantly less POC and PON (mol cell⁻¹) were found in −P PAR compared to −P PAB, and the opposite radiation effect was observed in −N. Our study shows that despite interactive effects on some of the variables studied, N. spumigena tolerate high ambient UVR also under nutrient limiting conditions and maintain positive growth rate even under severe phosphorus limitation.     

Buffering of Segmental and Chromosomal Aneuploidies in Drosophila melanogaster

Chromosomal instability, which involves the deletion and duplication of chromosomes or chromosome parts, is a common feature of cancers, and deficiency screens are commonly used to detect genes involved in various biological pathways. However, despite their importance, the effects of deficiencies, duplications, and chromosome losses on the regulation of whole chromosomes and large chromosome domains are largely unknown. Therefore, to explore these effects, we examined expression patterns of genes in several Drosophila deficiency hemizygotes and a duplication hemizygote using microarrays. The results indicate that genes expressed in deficiency hemizygotes are significantly buffered, and that the buffering effect is general rather than being mainly mediated by feedback regulation of individual genes. In addition, differentially expressed genes in haploid condition appear to be generally more strongly buffered than ubiquitously expressed genes in haploid condition, but, among genes present in triploid condition, ubiquitously expressed genes are generally more strongly buffered than differentially expressed genes. Furthermore, we show that the 4^th chromosome is compensated in response to dose differences. Our results suggest general mechanisms have evolved that stimulate or repress gene expression of aneuploid regions as appropriate, and on the 4^th chromosome of Drosophila this compensation is mediated by Painting of Fourth (POF).     

The D816V Mutation of c-Kit Circumvents a Requirement for Src Family Kinases in c-Kit Signal Transduction

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis, and gain-of-function mutations of the receptor are frequently seen in several malignancies, including acute myeloid leukemia, gastrointestinal stromal tumors, and testicular carcinoma. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V), leading to constitutive activation of the receptor. In this study, we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as of activation of downstream signaling pathways including receptor ubiquitination and the Ras/Mek/Erk pathway. Our data demonstrate that, unlike wild-type c-Kit, the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition, we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. We further present evidence that, in contrast to wild-type c-Kit, Src family kinases are dispensable for c-Kit/D816V cell survival, proliferation, and colony formation. Taken together, we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival, thereby contributing to the transforming potential of c-Kit/D816V.The receptor for stem cell factor (SCF),² c-Kit, is a type III receptor tyrosine kinase that belongs to the same subfamily as the platelet-derived growth factor receptors, the Flt3 receptor, and the macrophage colony-stimulating factor receptor (1). The c-Kit gene is identical to the white spotting locus (W) in the mouse. c-Kit is expressed in the hematopoietic system, in the gastrointestinal system, in melanocytes, and in germ cells, and therefore loss-of-function mutations in c-Kit lead to defects in hematopoiesis, melanogenesis, and gametogenesis. Stimulation of the c-Kit receptor with its ligand, SCF, leads to receptor dimerization and activation of its intrinsic tyrosine kinase activity. Specific tyrosine residues are autophosphorylated, which results in the activation of downstream signaling pathways, including the Ras/Erk pathway and the PI3-kinase pathway (for review, see Ref. 2).One of the crucial steps in oncogenic transformation of cells is the gain of independence of external growth stimuli. This can be achieved in several different ways, including mutations that render receptor tyrosine kinases constitutively active in the absence of ligand stimulation. In the case of c-Kit, these mutations most commonly occur either in exon 11 (encoding the juxtamembrane region) and are found predominantly in gastrointestinal stromal tumors or in exon 17 (encoding the activation loop of the kinase domain). A frequently occurring type of mutation in exon 17 in c-Kit is at codon 816. This type of mutation has been found in several human malignancies including acute myeloid leukemia, mastocytosis, germ cell tumors of the seminoma or dysgerminoma types, sinonasal natural killer/T-cell lymphomas, and in intracranial teratomas (3–10). These mutations at codon 816 lead to conversion of an aspartic acid residue to a valine, a tyrosine, a phenylalanine, an asparagine, or a histidine residue. The recently elucidated crystal structure of the c-Kit kinase domain has helped define the mechanism of activation by this type of mutation (11). In the unstimulated wild-type c-Kit, the juxtamembrane region inserts directly into the clefts between the amino- and carboxyl-terminal lobes of the kinase domain, disrupting the c-Kit control helix, and physically blocking the conserved kinase DFG motif from attaining a productive conformation. The activation loop folds back over the substrate binding groove and interacts with the active center of the kinase as a pseudosubstrate. It is not fully known how mutation of aspartic acid 816 leads to activation of c-Kit. It has been suggested either that the mutation inverts the conformation of the protein backbone so that the side chain of arginine 815 is being flipped from its position in the autoinhibited or that the effect of aspartic acid 816 mutations may be derived from its ability to stabilize the small positively charge α-helical dipole through its negative charge of its side chain. Asp-816 mutations in c-Kit promote receptor autophosphorylation and thereby constitutively activate downstream signaling pathways independent of SCF binding and therefore contribute to cell transformation (12). Imatinib (Gleevec) is a well known inhibitor of c-Kit juxtamembrane mutations and has been used in the treatment of gastrointestinal stromal tumors with activating mutations in the juxtamembrane region of c-Kit. In contrast, cells expressing c-Kit/D816V are resistant to Imatinib, whereas the Abl/Src dual inhibitor Dasatinib also inhibits the D816V mutant of c-Kit (13).Negative regulation of c-Kit signaling has been shown to occur mainly through ubiquitin-mediated internalization and degradation of the receptor (14, 15). Ubiquitination is mediated by ubiquitin E3 ligases that attach ubiquitin to their target proteins, resulting in either monoubiquitination or polyubiquitination. Key components in this machinery are the Cbl family of ubiquitin E3 ligases, represented by Cbl, Cbl-b, and Cbl-c (16). Signaling through receptor tyrosine kinases must be tightly regulated, and inhibition of Cbl activation and receptor ubiquitination can lead to cell transformation (17). It has been shown that both direct and indirect binding of Cbl to wild-type c-Kit can induce Cbl activation and receptor ubiquitination followed by receptor internalization and degradation (15, 18). In contrast, the mechanisms behind negative regulation of the oncogenic c-Kit/D816V are so far unknown.Extracellular signal-regulated kinase (Erk) proteins are the evolutionary conserved products of the two genes, Erk1 and Erk2, and are central proteins in the Ras/Erk signaling pathway. Erk1 and Erk2 are activated by dual phosphorylation on their regulatory tyrosine and threonine residues (19). The serine/threonine kinase Akt, also known as protein kinase B, is activated downstream of PI3-kinase and plays a central role in signaling induced by growth factors, cytokines, and other cellular stimuli (20). The Ras/Erk pathway and the PI3-kinase pathway are key signaling pathways involved in the regulation of cell proliferation, survival, and differentiation induced by c-Kit. A key player in the relay of signals from c-Kit into the cells is Src. Binding of Src to Tyr-568 in c-Kit leads to its activation and subsequent phosphorylation of Shc and activation of the Ras/Erk pathway (21). PI3-kinase is activated by c-Kit through two alternate routes, either through direct binding to Tyr-721 in c-Kit (22) or through indirect binding to the scaffolding protein Gab2, which associates to c-Kit via the adapter protein Grb2. Activation of PI3-kinase is dependent on Src-mediated phosphorylation of Gab2 (23). Other investigators have shown that neither Erk nor Akt is constitutively activated in cells expressing c-Kit/D816V (24).In this report, we demonstrate that in c-Kit/D816V-expressing Ba/F3 cells, a low constitutive activation of both Erk and Akt exists and that this activation can be further augmented by SCF stimulation. We also present data showing that c-Kit/D816V evades the need of Src family kinases for receptor ubiquitination and Erk activation by having an altered substrate specificity resembling Src family kinases. We conclude that Src family kinases play different roles in wild-type c-Kit and c-Kit/D816V-induced cell survival and growth.     

Cell guidance by magnetic nanowires

The phenomenon of contact guidance on thin fibers has been known since the beginning of the 20th century when Harrison studied cells growing on fibers from spider's web. Since then many studies have been performed on structured surfaces and fibers. Here we present a new way to induce guidance of cells or cell processes using magnetic nanowires. We have manufactured magnetic Ni-nanowires (200 nm in diameter and 40 μm long) with a template-based electro-deposition method. Drops of a nanowire/ethanol suspension were placed on glass cover slips. The nanowires were aligned in an external magnetic field and adhered to the cover slips after evaporation of the ethanol. When the wires had adhered, the magnetic field was removed. L929 fibroblasts and dissociated dorsal root ganglia (DRG) neurons from mice were cultured on the nanowire-coated cover slips for 24 h and 72 h respectively. The fibroblasts were affected by the aligned nanowires and displayed contact guidance. Regenerated axons also displayed contact guidance on the wires. There were no overt signs of toxicity caused by Ni-wires. Aligned magnetic nanowires can be useful for lab-on-a-chip devices and medical nerve grafts.