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Dictyosteliumdiscoideum (DD), an extensively studied model organism for cell and developmental biology, belongs to the most derived group 4 of social amoebas, a clade of altruistic multicellular organisms. To understand genome evolution over long time periods and the genetic basis of social evolution, we sequenced the genomes of Dictyostelium fasciculatum (DF) and Polysphondylium pallidum (PP), which represent the early diverging groups 1 and 2, respectively. In contrast to DD, PP and DF have conventional telomere organization and strongly reduced numbers of transposable elements. The number of protein-coding genes is similar between species, but only half of them comprise an identifiable set of orthologous genes. In general, genes involved in primary metabolism, cytoskeletal functions and signal transduction are conserved, while genes involved in secondary metabolism, export, and signal perception underwent large differential gene family expansions. This most likely signifies involvement of the conserved set in core cell and developmental mechanisms, and of the diverged set in niche- and species-specific adaptations for defense and food, mate, and kin selection. Phylogenetic dating using a concatenated data set and extensive loss of synteny indicate that DF, PP, and DD split from their last common ancestor at least 0.6 billion years ago.

Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. Investigation of a 48 h time course of infection revealed several clusters of co-regulated genes, an enrichment of preferentially up- or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co-incubation with Legionella hackeliae and L. pneumophilaDeltadotA. One hundred and thirty-one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host-pathogen interactions and for further studies on the Dictyostelium response to Legionella infection.

The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.

Eukaryotic cells contain a large number of actin binding proteins of different functions, locations and concentrations. They bind either to monomeric actin (G-actin) or to actin filaments (F-actin) and thus regulate the dynamic rearrangement of the actin cytoskeleton. The Dictyostelium discoideum genome harbors representatives of all G-actin binding proteins including actobindin, twinfilin, and profilin. A phylogenetic analysis of all profilins suggests that two distinguishable groups emerged very early in evolution and comprise either vertebrate and viral profilins or profilins from all other organisms. The newly discovered profilin III isoform in D. discoideum shows all functions that are typical for a profilin. However, the concentration of the third isoform in wild type cells reaches only about 0.5% of total profilin. In a yeast-2-hybrid assay profilin III was found to bind specifically to the proline-rich region of the cytoskeleton-associated vasodilator-stimulated phosphoprotein (VASP). Immunolocalization studies showed similar to VASP the profilin III isoform in filopodia and an enrichment at their tips. Cells lacking the profilin III isoform show defects in cell motility during chemotaxis. The low abundance and the specific interaction with VASP argue against a significant actin sequestering function of the profilin III isoform.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Enaptin belongs to a family of recently identified giant proteins that associate with the F-actin cytoskeleton as well as the nuclear membrane. It is composed of an N-terminal alpha-actinin type actin-binding domain (ABD) followed by a long coiled coil rod and a transmembrane domain at the C-terminus. The ABD binds to F-actin in vivo and in vitro and leads to bundle formation. The human Enaptin gene spreads over 515 kb and gives rise to several splicing isoforms (Nesprin-1, Myne-1, Syne-1, CPG2). The longest assembled cDNA encompasses 27,669 bp and predicts a 1014 kDa protein. Antibodies against the ABD of Enaptin localise the protein at F-actin-rich structures throughout the cell and in focal contacts as well as at the nuclear envelope. In COS7 cells, the protein is also present within the nuclear compartment. With the discovery of the actin-binding properties of Enaptin and the highly homologous Nuance, we define a family of proteins that integrate the cytoskeleton with the nucleoskeleton.

The viability of porcine collagenase-prepared islet preparations (n = 16) was classified by 31P-NMR spectroscopy, staining by neutral red and trypan blue, and in vitro insulin secretion following glucose challenge. Vital islets exhibited a phosphate diester/phosphate monoester (PDE/PME) ratio of 0.5-0.9, a staining score of 18-30 and an insulin secretion responding well to glucose challenge. Damaged islets performed at a PDE/PME of 0.2-0.49 and a staining score of 9-17 and necrotic islets had 0.0-0.49 and a staining score of 9-17 and necrotic islets had 0.0-0.19 and 0-8, respectively. The islets of the latter two groups did not adequately respond to glucose. The in vivo function following autotransplantation of these islets into the spleen was investigated in five recipients of more than 3000/kg vital islets of which 4 expressed daily normoglycemia (200 mg%), normalized intravenous glucose tolerance (K = -2.21), and a prolonged survival (mean +/- SD) of 167 +/- 12 days compared to five recipients of3000/kg damaged islets (K = -0.814) (P = 0.0017) and a survival of 86 +/- 21 days (P = 0.0096). It is suggested that 31P-NMR spectroscopy is a valuable and practical method to predict islet graft viability prior to transplantation in order to assure good graft function in the recipient.

The quality of pancreatic islets prepared by an intraductal pancreas collagenase perfusion technique was tested using three independent methods: 31P NMR spectroscopy, an insulin secretion test, and a staining method. The viability of pancreatic islet tissue was evaluated using the ratio of phosphate diester to phosphate monoester (PDE/PME) as a new criterion obtained by 31P NMR spectroscopy. According to this criterion, three types of tissue fragments could be characterized: vital (PDE/PME 0.5-0.9), damaged (PDE/PME less than 0.2), and necrotic (no PDE, no PME). The findings in the three different groups could be correlated to three trends of insulin secretion of the preparations following glucose challenge: good response to the glucose challenge, continuous decrease of insulin production, and no insulin secretion. We feel that 31P NMR spectroscopy offers a rapid and suitable method for classifying the viability of isolated pancreatic islets.

Im Experiment wurde der Einflus der SPV auf den Phosphatmetabolismus der Magenwand bei Ratten mittels 31P-NMR Spektroskopie untersucht. Die SPV bewirkt unmittelbar nach der Operation eine Destruktion des Stoffwechsels der Magenwandzellen. Vier Tage nch der SPV kommt es zur Regeneration des Metabolismus. Eine Woche nach der Operation ist die funktionelle Zellregeneration abgeschlossen, der Stoffwechsel aber verlangsamt. Dies hat eine reduzierte Produktion von S`aure und Schutzfaktoren der Magenschleimhaut zur Folge. Experimentelle Laasionen der Magenschleimhaut durch Phenylbutazon bewirken ebenso eine Zerstorung des Stoffwechsels der Magenwandzellen bis zum kompletten Zelluntergang.

The metabolism of choline in rabbit brain was studied by the noninvasive approach of in vivo13C NMR spectroscopy. 13C-Enriched precursors were introduced into the brain. Surgery of the head skin was avoided through controlled localization of the surface coil. For long-term accumulation studies in brain, repeated subcutaneous injections proved to be advantageous over other forms of application. The resorption kinetics was calculated to be zero order which suggests slow delivery from the subcutaneous depots. Choline metabolism was studied by two approaches: [N-13CH3] choline and S-[ 13CH3 ] methionine were administered separately to adult and myelinating rabbits (Days 5 to 32), respectively, over 4 weeks. [ N-13CH3] Choline and the 13CH3 group of methionine were incorporated into lecithin and sphingomyelin of brain myelin. In vivo kinetic studies of the turnover of these labeled structures were carried out. Choline and methionine are readily transported through the blood-brain barrier and utilized by the myelinating brain for the biosynthesis of its phospholipids.

The mobility of the lipids in the bilayer of the envelope of vesicular stomatitis virus has been probed over its complete space by the biosynthetic incorporation of [N-13CH3]- choline as a probe for the polar head groups and [3-13C]- and [11-13C] oleic acid and [16-13C]- palmitic acid for the hydrophobic region of the bilayer. These precursors were effectively incorporated as established by the concomitant administration of the same precursors in radioactive form. Spin lattice relaxation time measurements (T1) of the 13C enriched segments in complete virus envelope allowed estimation of their mobility. The mobility of the polar head groups is restricted, probably due to ionic interactions with neighbouring acidic phospholipids (phosphatidylserine) and/or acidic side chains of the glycoprotein (G-protein). The rigidity of the hydrophobic part of the bilayer is due to the high cholesterol content and interaction with the immersing polypeptide chains of the G- and possibly M-protein. The rigidity is limited to a depth of about 15 A ranging from the inner and outer surface, whereas the inner core of the bilayer is fluid. Tryptic cleavage of the hydrophilic part of the G-protein allows the lipophilic immersing polypeptide fragment to enter further the bilayer which then reduces the fluidity of the hydrocarbon chains in the core region by lipid-protein interactions.

Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.

Phosphatidylcholines, sphingomyelins, cholesterol, and cholesterol esters were enriched with 13 C by chemical synthesis in specific positions of their hydrophilic groups and aliphatic chains. Their spin-lattice relaxation times were determined in organic solvents. The substances were organized as liposomes and recombined with total human high density apolipoproteins and the two separated main components, apolipoprotein A-I (apoLp-Gln-I) and apolipoprotein A-II (apoLp-Gln-II). These 13 C nuclear magnetic resonance data established that in reassembled high density lipoproteins the phospholipid molecules bind to the apoprotein moieties with their hydrophobic fatty acid chains and not with their hydrophilic zwitterionic groups. Apolipoprotein A-I preferentially binds phosphatidylcholine, although its lipid-binding capacity is smaller than that of apolipoprotein A-II. Apolipoprotein A-II avidly reassembles with sphingomyelin by hydrophobic interactions. A model of the molecular organization of the high density lipoportein particle has been derived.

Die Bestrahlung der Phosphonodiazomethane 4 a – d (dargestellt durch Bamford-Stevens-Reaktion) und 6 (dargestellt durch Diazogruppen-Ubertragung) in Benzol liefert 7-Phosphono-7-aryl-norcaradiene (7 a – e) sowie 3.8-Bis-phosphono-3.8-diaryl-tricyclo[5.1.0.02.4]octene-(5) (9a – e). Fur 7 a wird ein temperaturabhangiges Valenzisomerie-Gleichgewicht gemas 7 ⇇ 8 NMR-spektroskopisch nachgewiesen. Wahrend p-Xylol mit photolytisch erzeugtem Dimethylphosphono-phenyl-carben ausschlieslich H-Abstraktionsprodukte liefert (16, 17, 18), reagieren p-Dichlor- und p-Dimethoxybenzol mit Phosphono-aryl-carbenen zu den 2.5-Dichlor- und 2.5-Dimethoxy-norcaradienen 19 a – d. Sie zeichnen sich durch ausergewohnliche thermische Stabilitat aus. Dies gilt auch fur die aus o-disubstituierten Benzolen auf dem Carbenwege erhaltenen Norcaradiene 21 a, b und 23. In allen Dreiringverbindungen ist die Phosphonogruppe offenbar exo-standig, wie die von Cyclopropan 32 abgeleitete 15 – 17 Hz-Kopplung von 31P mit cis-β-Wasserstoff anzeigt. Einige Norcaradiene wurden durch Diels-Alder-Addukte (34a – h) charakterisiert. Carbenes, V 7-Phosphono-7-arylnorcaradienes The photolysis of the phosphonodiazomethanes 4 a – d (prepared by Bamford-Stevens reaction) and 6 (prepared by diazo group transfer) in benzene yields 7-phosphono-7-arylnorcaradienes (7 a – e) as well as 3.8-diaryl-3.8-bis(phosphono)tricyclo[5.1.0.02.4]oct-5-enes (9 a – e). A temperature-dependent valence-isomeric equilibrium 7 ⇇ 8 is proved for 7 a by n. m. r. spectroscopy. p-Xylene reacts with photolytically generated (dimethylphosphono)-phenylcarbene exclusively with formation of H-abstraction products (16, 17, 18), whereas p-dichloro- and p-dimethoxybenzene react with (phosphono)arylcarbenes to yield the 2.5-dichloro- and 2.5-dimethoxynorcaradienes 19 a – d. These as well as the norcaradienes 21 a, b, and 23, formed from carbenes and o-disubstituted benzenes, possess an unusual thermal stability. The phosphono group apparently takes the exo-position in all cyclopropane compounds, as can be deduced from the 15 – 17 Hz coupling of 31P with cis-β-hydrogen in the cyclopropane 32. Some norcaradienes were characterized by Diels-Alder adducts (34a – h).

Es wird uber die Temperaturabhangigkeit des 1H-NMR-Spektrums von 4,5-Bis(methoxycarbonyl)-3,6-diphenyl-1 -p-tosyl-1H-azepin (11) berichtet. Die Befunde werden im Sinne einer Benzolimin-1H-Azepin-Valenztautomerie (Gleichgewichts-Verteilung bei Raumtemperatur ca. 3 : 97) interpretiert. Proton Resonance Spectroscopy of Unsaturated Ringsystems, XVIII.1 A Benzeneimine-1 H -Azepine Equilibrium The temperature dependence of the 1H n.m.r. spectrum of 4,5-bis(methoxycarbonyl)-3,6-diphenyl-1-p-tosyl-1H-azepine (11) is described. The results are interpreted on the basis of a benzeneimine-1H-azepine valence tautomerism (equilibrium distribution at room temperature ca. 3 : 97).

Publisher Summary This chapter discusses the studies on the regiospecificities in the binding of complex lipids. A thorough understanding of the molecular events in the function of lipid requiring proteins, for example, lipid transporting particles such as serum lipoproteins or intrinsic membrane proteins, demands the detailed knowledge of the molecular basis of the association of these specific apoproteins with their naturally occurring lipid classes and species or if model systems are being studied the lipid species should mimic as closely as possible the naturally associated ones. 13C-NMR-spectroscopy can measure two parameters accurately: the longitudinal relaxation time T1 and the nuclear overhauser enhancement (NOE). Their functional dependence on the correlation times is substantially different.

Glycophorin, the MN blood group substance, is a major intrinsic glycoprotein in erythrocyte membranes. The interaction of glycophorin with phosphatidylcholine, 13C-labeled in specific positions in reconstituted unilamellar vesicles, was investigated by using the 13C NMR technique. 1-Palmitoyl-2-([14-13C]linoleoyl)-sn-glycero-3-phosphocholine was synthesized and used as a probe. At 37 degrees C the spin-lattice relaxation time (T1) of vesicle bilayers consisting of this phospholipid was 0.74 s in the absence of glycophorin. The incorporation of glycophorin decreased the T1 to 0.63 s, indicating that the bulk lipid molecules are somewhat immobilized by glycophorin. In addition to the reduction in time, a broad component (delta H1/2 = approximately 40 Hz) superimposing the sharp resonance was observed in the 13C NMR spectrum of reconstituted vesicles. The T1 of the broad component was 0.32 s, suggesting that the lipid molecules contributing to the broad component may be more restricted than that of the sharp component. In order to quantify the broad component, a computer simulation was performed. The intensity of the broad component estimated from the simulation depended linearly on the concentration of glycophorin. Therefore, the broad component is considered to be contributed by a phospholipid domain surrounding the glycophorin molecules, a so-called "boundary lipid". The relationship between the broad component and the stoichiometry of the reconstituted vesicles allows the conclusion that about 30 lipid molecules are immobilized by one glycophorin monomer.

Native human serum high density lipoprotein (HDL) (d = 1.063--1.21g x cm-3) was enriched with phosphatidylcholines labelled with 13C in the polar head group ([N-13CH3]choline) and in the fatty acyl chains ([26-13C]cholesterol) and its linoleic acid ester using the previously described exchange method (Stoffel et al. 1978). The properties of the HDL particles with the exchanged lipid classes were the same as those of the native particles (Mr, CD, fluorescence, lipid and apoprotein stoichiometry, electrophoretic mobility). The T1-times were very similar to those obtained previously with recombined apolipoprotein-[13C]lipid complexes and further support our proposals concerning lipid and apoprotein interactions in the HDL particle.

Background Dictyostelium discoideum is frequently subjected to environmental changes in its natural habitat, the forest soil. In order to survive, the organism had to develop effective mechanisms to sense and respond to such changes. When cells are faced with a hypertonic environment a complex response is triggered. It starts with signal sensing and transduction and leads to changes in cell shape, the cytoskeleton, transport processes, metabolism and gene expression. Certain aspects of the Dictyostelium osmotic stress response have been elucidated, however, no comprehensive picture was available up to now. Results To better understand the D. discoideum response to hyperosmotic conditions, we performed gene expression profiling using DNA microarrays. The transcriptional profile of cells treated with 200 mM sorbitol during a 2-hour time course revealed a time-dependent induction or repression of 809 genes, more than 15% of the genes on the array, which peaked 45 to 60 minutes after the hyperosmotic shock. The differentially regulated genes were applied to cluster analysis and functional annotation using gene GO terms. Two main responses appear to be the down-regulation of the metabolic machinery and the up-regulation of the stress response system, including STATc. Further analysis of STATc revealed that it is a key regulator of the transcriptional response to hyperosmotic shock. Approximately 20% of the differentially regulated genes were dependent on the presence of STATc. Conclusion At least two signalling pathways are activated in Dictyostelium cells subjected to hypertonicity. STATc is responsible for the transcriptional changes of one of them.