Polysialylation of the neural cell adhesion molecule: interfering with polysialylation and migration in neuroblastoma cells

Polysialic acid represents a unique posttranslational modification of the neural cell adhesion molecule (NCAM). It is built as a homopolymer of up to 150 molecules of alpha 2-8-linked sialic acids on N-glycans of the fifth immunoglobulin-like domain of NCAM. Besides its role in cell migration and axonal growth during development, polysialic acids are closely related to tumor malignancy as they are linked to the malignant potential of several tumors, such as undifferentiated neuroblastoma. Polysialic acid expression is significantly more frequent in high-grade tumors than in low-grade tumors. It is synthesized in the Golgi apparatus by the activity of two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV. Interestingly, polysialylation of tumors is not equally synthesized by both polysialyltransferases. It has been shown that especially the ST8SiaII gene is not expressed in some normal tissue, but is strongly expressed in tumor tissue. Here we summarize some knowledge on the role of polysialic acid in cell migration and tumor progression and present novel evidence that interfering with polysialylation using unnatural sialic acid precursors decreases the migration of neuroblastoma cells.     

RNAi‐mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing

RNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O‐methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down‐regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3′ direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down‐regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit.     

Biochemical and physiological characterization of the pyruvate formate-lyase Pfl1 of Chlamydomonas reinhardtii, a typically bacterial enzyme in a eukaryotic alga

The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.     

Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy

It is shown that real-time 2D solid-state NMR can be used to obtain kinetic and structural information about the process of protein aggregation. In addition to the incorporation of kinetic information involving intermediate states, this approach can offer atom-specific resolution for all detectable species. The analysis was carried out using experimental data obtained during aggregation of the 10.4 kDa Crh protein, which has been shown to involve a partially unfolded intermediate state prior to aggregation. Based on a single real-time 2D ¹³C–¹³C transition spectrum, kinetic information about the refolding and aggregation step could be extracted. In addition, structural rearrangements associated with refolding are estimated and several different aggregation scenarios were compared to the experimental data.     

Recruitment of human cytomegalovirus immediate-early 2 protein onto parental viral genomes in association with ND10 in live-infected cells

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise co-localizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.     

S100A1 enhances the L-type Ca2+ current in embryonic mouse and neonatal rat ventricular cardiomyocytes

S100A1 is an EF-hand type Ca2+-binding protein with a muscle-specific expression pattern. The highest S100A1 protein levels are found in cardiomyocytes, and it is expressed already at day 8 in the heart during embryonic development. Since S100A1 is known to be involved in the regulation of Ca2+ homeostasis, we tested whether extracellular S100A1 plays a role in regulating the L-type Ca2+ current (I(Ca)) in ventricular cardiomyocytes. Murine embryonic (day 16.5 postcoitum) ventricular cardiomyocytes were incubated with S100A1 (0.001-10 microM) for different time periods (20 min to 48 h). I(Ca) density was found to be significantly increased as early as 20 min (from -10.8 +/- 1 pA/pF, n = 18, to -22.9 +/- 1.4 pA/pF; +112.5 +/- 13%, n = 9, p < 0.001) after the addition of S100A1 (1 microM). S100A1 also enhanced I(Ca) current density in neonatal rat cardiomyocytes. Fluorescence and capacitance measurements evidenced a fast translocation of rhodamine-coupled S100A1 from the extracellular space into cardiomyocytes. S100A1 treatment did not affect cAMP levels. However, protein kinase inhibitor, a blocker of cAMP-dependent protein kinase A (PKA), abolished the S100A1-induced enhancement of I(Ca). Accordingly, measurements of PKA activity yielded a significant increase in S100A1-treated cardiomyocytes. In vitro reconstitution assays further demonstrated that S100A1 enhanced PKA activity. We conclude that the Ca2+-binding protein S100A1 augments transsarcolemmal Ca2+ influx via an increase of PKA activity in ventricular cardiomyocytes and hence represents an important regulator of cardiac function.     

A globin gene of ancient evolutionary origin in lower vertebrates: evidence for two distinct globin families in animals

Hemoglobin, myoglobin, neuroglobin, and cytoglobin are four types of vertebrate globins with distinct tissue distributions and functions. Here, we report the identification of a fifth and novel globin gene from fish and amphibians, which has apparently been lost in the evolution of higher vertebrates (Amniota). Because its function is presently unknown, we tentatively call it globin X (GbX). Globin X sequences were obtained from three fish species, the zebrafish Danio rerio, the goldfish Carassius auratus, and the pufferfish Tetraodon nigroviridis, and the clawed frog Silurana tropicalis. Globin X sequences are distinct from vertebrate hemoglobins, myoglobins, neuroglobins, and cytoglobins. Globin X displays the highest identity scores with neuroglobin (approximately 26% to 35%), although it is not a neuronal protein, as revealed by RT-PCR experiments on goldfish RNA from various tissues. The distal ligand-binding and the proximal heme-binding histidines (E7 and F8), as well as the conserved phenylalanine CD1 are present in the globin X sequences, but because of extensions at the N-terminal and C-terminal, the globin X proteins are longer than the typical eight alpha-helical globins and comprise about 200 amino acids. In addition to the conserved globin introns at helix positions B12.2 and G7.0, the globin X genes contain two introns in E10.2 and H10.0. The intron in E10.2 is shifted by 1 bp in respect to the vertebrate neuroglobin gene (E11.0), providing possible evidence for an intron sliding event. Phylogenetic analyses confirm an ancient evolutionary relationship of globin X with neuroglobin and suggest the existence of two distinct globin types in the last common ancestor of Protostomia and Deuterostomia.