Aphidicolin induces 6-thioguanine resistant mutants in human diploid fibroblasts

Cytotoxic and mutagenic effects of aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta, were studied in human diploid VH-10 fibroblasts. The cells were treated (2 or 4h) with APC at concentration ranges of 10-40 microM. The effect of APC on cell survival after 4 h treatment was significantly higher than after 2 h treatment. The mutagenicity of APC was investigated at the HPRT locus, and the frequency of HPRT mutants was estimated by selection in medium containing 6-thioguanine (6-TG). Treatment of fibroblast cells with 20 microM of APC for 2 or 4 h resulted approximately in 5 or 10 times increase of 6-TG resistant mutant frequencies, respectively, compared to untreated control cells.The cell cycle analyses performed during the expression time (9-12 days) have shown that after 2 and 4h treatment with APC the cells were blocked in G2 phase during the majority of the expression period, compared to control cells. Four days after the treatment, the amount of cells in G2 phase increased about two-fold (28.6-31.8% compared to 13.5% in the untreated cells). The mode of cell death during the expression time was via necrosis, rather than apoptosis, which was demonstrated by fluorescein-diacetate (FDA)-staining and terminal dUTP nick end labeling (TUNEL)-method.     

Genes associated with the end of dormancy in grapes

A grape bud EST library was constructed and 4,270 ESTs sequenced. The library clones were arrayed for the purpose of investigating the level of gene expression over time, particularly leading up to the buds release from dormancy. The arrays were hybridized with P³³-labeled probes produced from samples of buds collected at weekly intervals. These probes covered the time from 9 weeks prior to bud burst until just after the emergence of the shoots. Expression patterns from these genes have been examined. It was found that 74% of the genes in the data set were homologous to known proteins. Genes were then assigned to functional categories according to their primary BLAST match. Of these 13% were involved with photosynthesis, 13% with disease resistance and defense, 5% energy, 12% metabolism, 20% protein production and processing, 25% cell structure and plant growth and the remaining 12% were unclassified The expression pattern of a selection of candidate genes retrieved from literature previously reporting an association with dormancy changes was assessed. On closer examination most of these genes relate to the oxidative processes and stress responses within the cell. The results of this study show that even in the dormant state, gene expression in the buds is high.     

An unusual halotolerant alpha-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli

A 60-kDa, salt-inducible, internally duplicated alpha-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.     

New achievements in human cell toxicology: the 20th annual workshop on in vitro toxicology

The 20th Annual Workshop on In Vitro Toxicology (Oxford, UK, September 22-24, 2002) was convened as part of a European meeting entitled Human Cell Culture 2002. The meeting was arranged by the Scandinavian Society for Cell Toxicology (SSCT), the European Tissue Culture Society and the British Prostate Group. Two sessions, which are summarised in this report, were devoted to in vitro toxicology: Human Cell Toxicology and The SSCT Free Paper Session. Outstanding experts in the field of toxicology outlined contemporary approaches in toxicity testing in their lectures. Short oral presentations demonstrated a variety of in vitro model systems and methodologies, which can be useful for investigating human toxicity, as well as for studies on mechanisms of toxicity.     

Genetic Diversity and Spatial Genetic Structure of Chamaedaphne calyculata (Ericaceae) at the Western Periphery in Relation to its Main Continuous Range in Eurasia

A previous phylogeography and genetic diversity study of Chamaedaphne calyculata (Ericaceae) showed that populations over its geographic range were strongly separated into two groups: a Eurasian/NW North American group and a NE North American one corresponding with the disjunct distribution of Sphagnum-dominated peatlands in north-western and central-eastern North America. Here, I have extended the survey and focused on the species’ detailed postglacial origin and the effect of isolation on genetic diversity patterns, particularly within island-like populations at the western periphery of its range in Europe. Using AFLP markers, estimates of genetic diversity within 16 C. calyculata populations in the Eurasian group were low (percentage of polymorphic loci P _PL=14.9–24.8 %, Nei’s gene diversity H=0.060–0.119). Genetic diversity patterns within this species did not support the hypothesis that genetic diversity decreases towards the periphery of the range. Bayesian clustering analysis showed that population-level admixture was present in almost all studied 16 populations, suggesting multi-directional gene flow. On the other hand, the majority of assigned individuals (ca. 98 % of individuals) were offspring of the original residents, confirming that C. calyculata populations in the present day acted as discrete genetic units both in its continuous range and at its western periphery, and that gene flow was historic rather than contemporary in Eurasia. There was no correlation between genetic and geographic distance in the Eurasian group (r=0.02, P>0.05, Mantel test) nor at the western periphery (r=0.15, P>0.05, Mantel test). The isolation-by-distance (IBD) scatterplot matched Hutchinson and Templeton’s interpretation (case III), and geographic distance between populations was not a reliable predictor of the degree of genetic differentiation between populations. It is suggested that the lack of IBD might be a result of random genetic drift in rather disconnected populations that have become increasingly fragmented relatively recently. Positive and significant relationships between genetic and geographic distance on a small population scale was the result of biparental inbreeding of C. calyculata and restricted seed rain. Despite sporadic generative reproduction and limited dispersal, the fine-scale genetic structure within populations has been maintained, even though population sizes have been reduced to small fragments in recent years.     

IGF-1R tyrosine kinase expression and dependency in clones of IGF-1R knockout cells (R-)

Insulin-like growth factor 1 receptor (IGF-1R) plays many crucial roles in cancer, like anti-apoptotic activity and necessity for transformation. IGF-1R knockout cells (R-) represent a useful tool for molecular mapping of biological properties of the receptor. R- cells have been shown to be refractory to transformation by viral and cellular oncogenes, highlighting the necessity of this receptor for transformation. Surprisingly, more recent studies have shown that these cells can undergo spontaneous transformation. This observation raises the question as whether R- cells over the years have acquired some properties mimicking those of IGF-1R. Using an IGF-1R inhibitor (cyclolignan PPP) we have identified clones of R- (R-s) that are sensitive to this compound. Since, PPP is closely related to podophyllotoxin, which is an efficient microtubule inhibitor, we first investigated if such a mechanism could explain the sensitivity to PPP. However, highly purified PPP showed no or very slight tubulin binding. Further analysis of R-s revealed expression of a 90 kDa protein being reactive to IGF-1R beta-subunit antibodies. This protein was weakly but constitutively tyrosine phosphorylated and was downregulated by siRNA targeting IGF-1R. This downregulation was paralleled by decreased R-s survival. Taken together, our study suggests that clones of R- express IGF-1R activity and dependency, which in turn may explain that R- can undergo spontaneous transformation.     

In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (^·OH)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance spectroscopy to show that ^·OH is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum; Brassicaceae) seeds. Endosperm weakening precedes radicle emergence, as demonstrated by direct biomechanical measurements. By ³H fingerprinting, we showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic ^·OH in radicles and endosperm caps: the production and action of ^·OH increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of ^·OH attack on polysaccharides were evident. In vivo ^·OH attack on cell wall polysaccharides were evident not only in germinating seeds but also in elongating maize (Zea mays; Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by ^·OH is a controlled action of this type of reactive oxygen species.     

TNFA Haplotype Genetic Testing Improves HLA in Estimating the Risk of Celiac Disease in Children

Background

TNF-α and IFN-γ play a role in the development of mucosal damage in celiac disease (CD). Polymorphisms of TNFA and IFNG genes, as well as of the TNFRSF1A gene, encoding the TNF-α receptor 1, might underlie different inter-individual disease susceptibility over a common HLA risk background. The aims of this study were to ascertain whether five SNPs in the TNFA promoter (-1031T>C,-857C>T,-376G>A,-308G>A,-238G>A), sequence variants of the TNFRSF1A gene and IFNG +874A>T polymorphism are associated with CD in a HLA independent manner.

Methods

511 children (244 CD, 267 controls) were genotyped for HLA, TNFA and INFG (Real Time PCR). TNFRSF1A variants were studied (DHPLC and sequence).

Results

Only the rare TNFA-1031C (OR=0.65, 95% CI:0.44-0.95), -857T (OR=0.42, 95% CI:0.27-0.65), -376A (OR=2.25, 95% CI:1.12-4.51) and -308A (OR=4.76, 95% CI:3.12-7.26) alleles were significantly associated with CD. One TNFRSF1A variant was identified (c.625+10A>G, rs1800693), but not associated with CD. The CD-correlated TNFA SNPs resulted in six haplotypes. Two haplotypes were control-associated (CCGG and TTGG) and three were CD-associated (CCAG, TCGA and CCGA). The seventeen inferred haplotype combinations were grouped (A to E) based on their frequencies among CD. Binary logistic regression analysis documented a strong association between CD and HLA (OR for intermediate risk haplotypes=178; 95% CI:24-1317; OR for high risk haplotypes=2752; 95% CI:287-26387), but also an HLA-independent correlation between CD and TNFA haplotype combination groups. The CD risk for patients carrying an intermediate risk HLA haplotype could be sub-stratified by TNFA haplotype combinations.

Conclusion

TNFA promoter haplotypes associate with CD independently from HLA. We suggest that their evaluation might enhance the accuracy in estimating the CD genetic risk.