Phenotypic and molecular characterization of inducible human neuroblastoma cell lines

Two new neuroblastoma (NB) cell lines, NUB-6 and NUB-7, were established from recurrent and primary NB tumours respectively and identified conclusively as NB by their phenotypic characteristics, catecholamine production and N-myc amplification. The cell lines could be distinguished on the bases of distinctive growth patterns in monolayer culture and semi-solid media (collagen gel and agarose), neurite formation and their response to four classes of growth and differentiation modulators. The NUB-6 cell line consisted of two distinct cell subtypes, small typical neuroblasts and larger spheroid-forming cells, while NUB-7 was homogeneously neuroblastic. Class-I agents (dibutyrl cyclic AMP [dbcAMP], butyrate, and papaverine) inhibited growth of both cell lines, while only dbcAMP stimulated the formation of short neurites by NUB-6 neuroblast cells in monolayer culture and collagen. Of the class-II agents (vitamins), retinoic acid inhibited growth of both cell lines and stimulated formation of long neurites by NUB-6 cells and NUB-7 cells in later passages. In contrast, vitamin E inhibited growth of NUB-6 and late-passage NUB-7, but stimulated early passage NUB-7. The class III agent (nerve growth factor) resembled vitamin E. The class-IV agents (interferons; rIFN-alpha 2a and rIFN-gamma 1) inhibited growth of both cell lines in monolayer culture and agarose, but stimulated NUB-6 neuroblasts and early passage NUB-7 cells to form long neurites. Thus phenotypically distinct NB cell lines were established in vitro and shown to be differentially influenced by various growth and differentiation modulators. The potent effect of IFN suggests a role for these modulators in NB behaviour in vivo.     

The Plasmodium falciparum Ca(2+)-ATPase PfATP6: insensitive to artemisinin, but a potential drug target

The disease malaria, caused by the parasite Plasmodium falciparum, remains one of the most important causes of morbidity and mortality in sub-Saharan Africa. In the absence of an efficient vaccine, the medical treatment of malaria is dependent on the use of drugs. Since artemisinin is a powerful anti-malarial drug which has been proposed to target a particular Ca2+-ATPase (PfATP6) in the parasite, it has been important to characterize the molecular properties of this enzyme. PfATP6 is a 139?kDa protein composed of 1228 amino acids with a 39% overall identity with rabbit SERCA1a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a). PfATP6 conserves all sequences and motifs that are important for the function and/or structure of a SERCA, such as two high-affinity Ca2+-binding sites, a nucleotide-binding site and a phosphorylation site. We have been successful in isolating PfATP6 after heterologous expression in yeast and affinity chromatography in a pure, active and stable detergent-solubilized form. With this preparation, we have characterized and compared with the eukaryotic SERCA1a isoform the substrate (Ca2+ and ATP) -dependency for PfATP6 activity as well as the specific inhibition/interaction of the protein with drugs. Our data fully confirm that PfATP6 is a SERCA, but with a distinct pharmacological profile: compared with SERCA1a, it has a lower affinity for thapsigargin and much higher affinity for cyclopiazonic acid. On the other hand, we were not able to demonstrate any inhibition by artemisinin and were also not able to monitor any binding of the drug to the isolated enzyme. Thus it is unlikely that PfATP6 plays an important role as a target for artemisinin in the parasite P. falciparum.     

In vitro primary satellite cell growth and differentiation within litters of pigs

Postnatal muscle growth is dependent on satellite cell (SC) proliferation, differentiation and fusion to increase the DNA content of existing muscle fibres and thereby the capacity to synthesize protein. The purpose of the present study was to examine the ability of isolated SCs from low, medium and high weaning weight litter mates of pigs to proliferate and differentiate, and to affect protein synthesis and degradation after fusion into myotubes. At 6 weeks of age, SCs from the lowest weight (LW), medium weight (MW) and highest weight (HW) female pigs within eight litters were isolated. Thereby, eight cultures of SCs were established for each of the three weight groups within litter, representing three groups of SCs from pigs exhibiting differences in postnatal muscle growth performance. Proliferation was estimated as the number of viable cells at different time points after seeding. SC differentiation was evaluated by measuring the activity of the muscle-specific enzyme, creatine phosphokinase, and protein synthesis and degradation were measured by incorporation and release of 3H-tyrosine, respectively. A tendency towards a difference in proliferation between SC cultures was found (P = 0.09). This was evident as the number of viable cells at day 3 was lower in cultures from LW pigs than from HW (P < 0.05) and MW (P < 0.01) pigs. Differentiation was significantly different between cultures (P < 0.05). There was a significant difference between LW and MW cultures at 72 h (P < 0.05), and a tendency towards a difference between LW and HW cultures at 45 h (P = 0.07). Protein synthesis per μg protein or per μg DNA did not differ among SC cultures from LW, MW and HW pigs. Neither did protein degradation rate differ significantly among SC cultures from LW, MW and HW pigs. Overall, the results show that SCs from LW pigs seem to proliferate and differentiate at a slower rate than SCs from MW and HW pigs. The results found in this study show no difference in the ability of SCs to affect protein synthesis or degradation between SCs from litter mates exhibiting different growth rates in vivo.     

Quantification of Indole-3-Acetic Acid in Dark-Grown Seedlings of the Diageotropica and Epinastic Mutants of Tomato (Lycopersicon esculentum Mill.)

Endogenous indoleacetic acid (IAA) levels were examined in 7-day-old, dark-grown tomato seedlings (Lycopersicon esculentum Mill. cv VFN8), and in two single-gene mutants, Epinastic and diageotropica. Gas chromatography-mass spectrometry was employed to quantify IAA using ¹³C₆-[benzene ring]indoleacetic acid as internal standard. IAA concentrations ranged from 89 to 134 nanograms per gram dry weight and were not significantly different for the three genotypes. Ethylene over-production by dark-grown Epi seedlings is not likely to result from increased IAA. Assuming similar recovery percentages for each genotype, indole-3-ethanol, a purported storage form of IAA, was identified by GC-MS and found to be more prevalent in the parent tomato, VFN8, with only trace amounts observed in Epi. No IEt was detected by high performance liquid chromatography/fluorescence in dgt (detection limit >100 picograms).     

Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.      

Safety evaluation of sous vide-processed ready meals

AIMS: To assess survival, growth and toxin production of spore-forming bacteria in sous vide products exposed to a relatively high heat treatment. METHODS AND RESULTS: During a three-year period, 2,168 sous vide-processed, commercially available ready-made meals with a shelf life of 3-5 weeks were examined. The products were stored at 4 degrees C for the first 1/3 and at 7 degrees C for the remaining 2/3 of their shelf life period. Three-fourths of the samples had less than 10 bacteria per gram the day after production, and none had more than 1,000. Similar numbers were found at the end of the shelf life when stored as described above. At abuse temperature (20 degrees C), the number of bacteria increased to 10(6)-10(7) cfu g(-1) 7 d after production. A total of 350 isolates of Bacillus spp. were collected, but no Clostridium strains were detected. Only 11 of the 113 tested strains were able to grow at 7 degrees C in broth, and none of the psychrotrophic strains were able to produce substantial amounts of toxins causing food poisoning. CONCLUSION: The health risk of these products is small as long as the temperature during storage is low. For microbial testing of the end products, traditional plating will suffice.     

Splenocytes cultured in low concentrations of IL-2 generate NK cell specificities toward syngenic and allogenic targets

Splenocytes cultured in the presence of 30-60 units/ml IL-2 for 5 days develop natural killer activity toward syngeneic and allogeneic tumor cell targets. The IL-2 activated splenocytes, themselves, are partially resistant, whereas concanavalin A-activated T blast cells are completely resistant to killing. Surprisingly, major histocompatibility complex (MHC)-I-negative target cells are also resistant to natural killer (NK)-cell-mediated killing. Cells resistant to killing were unable to block NK-cell-mediated killing of sensitive targets as judged from cold target cell inhibition experiments, and one type of target cells sensitive to killing did generally not cross-block killing of other killing-sensitive target cell types. Alloantigen exposure of splenocytes, i.e., one-way mixed lymphocyte cultures, partially prevents the development of NK-cell activity. Our data suggest that target structures which trigger killing activity of NK cells are determined by the phenotype of the target cell and are dependent on its MHC class I expression disregarding the haplotype of the cell.     

Validity of the multiphasic concept of ion absorption in plants

Borstlap (Naturwissenschaften 68: 41–43, 1981; Plant Cell Environ. 4: 189–195, 1981) has alleged that the concept of multiphasic uptake of solutes in plants is invalid. This claim is considered in detail and shown not to be justified. Contrary to Borstlap, the concentration-dependence of uptake cannot be adequately represented by the sum of two Michaelis-Menten terms and a linear term, or by similar continuous functions, if sufficiently detailed and precise data are used. Published evidence for the multiphasic concept, including the existence of discontinuities and separate uptake and transition sites, remains valid.     

Bacteria-mediated uptake of choline sulfate by plants. Bacterial effectiveness

The ability of bacteria to cause rapid uptake of choline sulfate in plants, i.e., effectiveness, was studied using Pseudomonas tolaasii and excised roots of barley (Hordeum vulgare L.). Once effective, bacteria remained so after being killed by treatments which cause little damage to their outer structure. However, effectiveness was destroyed by disruption of the cell wall, protein reagents, a mild heat treatment or removal of Mg²⁺. Effective bacteria adsorbed choline sulfate. This adsorption had characteristics similar to those of bacterial effectiveness (magnesium requirement, high substrate specificity). These results indicate that a proteinaceous structure on the bacterial surface binds and, somehow, transfers choline sulfate to the plant.