Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.     

Characterization of a porcine glucosephosphate isomerase-processed pseudogene at Chromosome 1q1.6-1.7

A porcine glucosephosphate isomeraseprocessed pseudogene has been isolated and sequenced. The pseudogene has several base substitutions as well as an insertion and deletions, and is 83% homologous to the corresponding cDNA. It contains an intervening sequence of 565 bp, is truncated at the 3 end, and is flanked by direct repeats of seven nucleotides. Fluorescent in situ hybridization to porcine metaphase chromosomes localized the processed pseudogene to Chromosome (Chr) 1q1.6-1.7. A (GT)₁₄(AT)₁₅ microsatellite was detected close to the processed pseudogene.     

Enhancement of DNA-mediated gene transfer by inhibitors of autophagic-lysosomal function

A variety of compounds, known to influence the intravesicular transport and degradation of macromolecules, was studied for their effect on the efficiency of DNA-mediated gene transfer (transfection). The efficiency of transfection was measured by transformation of rat 2 thymidine kinase-deficient (tk^?) cells by the cloned herpes simplex I thymidine kinase gene (pAGO). When salmon sperm DNA (average molecular weight, 6 × 10⁶ D) was used as a carrier, the presence of either 20 mM NH₄Cl, 1 μM carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), or 5 mM 3-methyl adenine (3-MA) in the medium during incubation of the cells with the DNA-calcium-phosphate (DNA-Ca-P_i) precipitate, enhanced the efficiency of transfection by a factor of 10. If rat thymus DNA (greater than 30 × 10⁶ D) was used as a carrier, the transformation efficiency was much higher than with salmon sperm DNA. However, in this case treatment with 3-MA, NH₄Cl and FCCP enhanced the transformation frequency by slightly less than a factor of two. 3-MA further increased the transfection frequency if the cells were incubated with the compound after removal of the DNA-Ca-P_i coprecipitate, whereas NH₄Cl and FCCP had no such effect. Our results strongly suggest that these inhibitors of intracellular degradation can increase the frequency of transformation by increasing the cytoplasmic levels of exogenous DNA.     

Substrate-enzyme interactions and catalytic mechanism in phospholipase C: a molecular modeling study using the GRID program.

Based on the high-resolution X-ray crystallographic structure of phospholipase C from Bacillus cereus, the orientation of the phosphatidylcholine substrate in the active site of the enzyme is proposed. The proposal is based on extensive calculations using the GRID program and molecular mechanics geometry relaxations. The substrate model has been constructed by successively placing phosphate, choline and diacylglycerol moieties in the positions indicated from GRID calculations. On the basis of the resulting orientation of a complete phosphatidylcholine molecule, we propose a mechanism for the hydrolysis of the substrate.     

Causes and characteristics of reverse bird migration: an analysis based on radar,radio tracking and ringing at Falsterbo,Sweden

That birds migrate in the reverse direction of the expected is a phenomenon of regular occurrence which has been observed at many sites. Here we use three different methods; tracking radar, radiotelemetry and ringing, to characterize the flights of these reverse migrants and investigate possible causes of reverse migration of nocturnally migrating passerines during autumn migration at Falsterbo peninsula, Sweden. Using these different methods we investigated both internal factors, such as age and fuel load, and external factors such as weather variables, competition and predation risk. Birds flying in the reverse direction were more likely to be lean and to be juveniles. Reverse migration was also more common with overcast skies and winds with north and east components. We did not find any effect of temperature, visibility, number of migrating sparrowhawks, or the total number of ringed birds at the site on the day of departure. We found that reverse migration is characterized by slower flight speeds (airspeed) at high altitudes and that it takes place later in the night than forward migration.     

Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.     

How specific MHC class I and class II combinations affect disease resistance against infectious salmon anaemia in Atlantic salmon (Salmo salar)

The aim was to evaluate the performance of selected individual MHC class I and class II alpha (A) alleles, and combinations of these on disease resistance against infectious salmon anaemia (ISA). The material consisting of 1966 fish from seven families, contained five MHC class I alleles and four MHC class II A alleles. Which representing given class II A and class II beta (B) haplotypes, totalling 19 MHC class I and class II A genotypes. The fish were challenged with infectious salmon anaemia virus (ISAV), the virus causing ISA. Dead fish were collected daily during the challenge experiment and the survivors were collected at termination. All fish were genotyped for MHC class I and class II A. The total mortality in the material was 85.14%. For MHC class I, UBA*0201 and UBA*0301 were significantly the most resistant alleles, while UBA*0601 for class I and DAA*0301 for class II A were the significantly most susceptible alleles. The analysis of combined MHC class I and class II A genotypes detected that fish with the genotype UBA*0201/*0301;DAA*0201/*0201 were the most resistant fish with a hazard ratio (HR) at 0.750, while the fish with the genotypes UBA*0601/*0801;DAA*0501/*0501 and UBA*0201/*0301;DAA*0301/*0501 were the most susceptible fish with HR of 1.334 and 1.425. In addition, Cox regression analysis within family detected combined MHC class I and class II A genotypes that contributed significantly to resistance and susceptibility. The study confirmed the expectation of performance of individual MHC class I and class II A alleles, and also detected an effect of MHC class I and class II A in combinations.     

GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

Alpha-helix formation is required for high affinity binding of human apolipoprotein A-I to lipids

Apolipoprotein (apo) A-I is thought to undergo a conformational change during lipid association that results in the transition of random coil to alpha-helix. Using a series of deletion mutants lacking different regions along the molecule, we examined the contribution of alpha-helix formation in apoA-I to the binding to egg phosphatidylcholine (PC) small unilamellar vesicles (SUV). Binding isotherms determined by gel filtration showed that apoA-I binds to SUV with high affinity and deletions in the C-terminal region markedly decrease the affinity. Circular dichroism measurements demonstrated that binding to SUV led to an increase in alpha-helix content, but the helix content was somewhat less than in reconstituted discoidal PC.apoA-I complexes for all apoA-I variants, suggesting that the helical structure of apoA-I on SUV is different from that in discs. Isothermal titration calorimetry showed that the binding of apoA-I to SUV is accompanied by a large exothermic heat and deletions in the C-terminal regions greatly decrease the heat. Analysis of the rate of release of heat on binding, as well as the kinetics of quenching of tryptophan fluorescence by brominated PC, indicated that the opening of the N-terminal helix bundle is a rate-limiting step in apoA-I binding to the SUV surface. Significantly, the correlation of thermodynamic parameters of binding with the increase in the number of helical residues revealed that the contribution of alpha-helix formation upon lipid binding to the enthalpy and the free energy of the binding of apoA-I is -1.1 and -0.04 kcal/mol per residue, respectively. These results indicate that alpha-helix formation, especially in the C-terminal regions, provides the energetic source for high affinity binding of apoA-I to lipids.