The genome of the social amoeba Dictyostelium discoideum.

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.

In vitro and in vivo viability assessment of unpurified pancreatic islet tissue.

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 of > 3000/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 pig--an experimental model for transplantation of isolated islets pancreatic islets]. / Osípaná--experimentálny model pre transplantáciu izolovaných ostrovcekov pankreasu.

The authors present results of autotransplantation of the islets of Langerhans of the pancreas in pigs and miniature pigs ("Troll"). Diabetes was induced by total pancreatomy. From the removed pancreas the islets of Langerhans were separated by the method of intraductal perfusion with collagenase and implanted into the spleen or liver via the portal vein. The mortality on operation in pigs was 33%, in miniature pigs 20%. In domestic pigs normal blood sugar levels were achieved in 25% after implantation of the islets of Langerhans into the liver (in 2 of 8 animals) and in 50% after implantation into the spleen (in 4 of 8 animals). In miniature pigs autotransplantation of the islets of Langerhans was equally successful when transplanted into the liver or the spleen -50%. The 50% success of autotransplantations justifies the use of pigs as a model for further investigations of allo- or xenotransplantations of the islets of Langerhans.

31P NMR spectroscopy for in vitro viability testing of porcine pancreatic islets.

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.

[31P nuclear magnetic resonance spectroscopy of the stomach wall following proximal selective vagotomy]. / 31P Kernspinspektroskopie der Magenwand nach proximaler selektiver Vagotomie.

The phosphate metabolism of the gastric wall after proximal selective vagotomy (PSV) was investigated by means of 31P-NMR spectroscopy. The destruction of metabolism has been found just after PSV resulting in a significant decrease of adenosine diphosphates and adenosine triphosphates. 4 and 7 days after PSV the progress of metabolism regeneration was detected, nevertheless with the retardation of high energy phosphates ischemic degradation. The results indicate not only a hydrochloric acid activity reduction but diminution of gastric mucosa protective factors, too. In experimental gastric ulcers no energy phosphates have been found suggesting a mucosa cells necrobiosis.

In vivo 13C nuclear magnetic resonance investigations of choline metabolism in rabbit brain.

The metabolism of choline in rabbit brain was studied by the noninvasive approach of in vivo 13C 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.

Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved.

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.

Carbon-13 nuclear magnetic resonance studies on the interaction of glycophorin with lecithin in reconstituted vesicles.

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.

13C-NMR spectroscopy of human serum high density lipoprotein enriched with labelled phospholipids.

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.

13C-NMR studies of the membrane structure of enveloped virions (vesicular stomatitis virus).

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.

13C nuclear magnetic resonance spectroscopic evidence for hydrophobic lipid-protein interactions in human high density lipoproteins.

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.