Rat embryo fibroblasts transformed by c-Jun display highly metastatic and angiogenic activities in vivo and deregulate gene expression of both angiogenic and antiangiogenic factors.

The comparative tumorigenicity in rats and nude mice of cell lines derived from FR3T3 and transformed by either c-jun, ras, SV40 lt, or bovine papilloma virus type 1 (BPV1) oncogenes was investigated. c-Jun-transformed cells were as tumorigenic and metastatic as Ras-transformed cells. Latencies were short, and numerous pulmonary metastases were observed in all injected animals. In contrast, tumors induced by s.c. injection of SV40-transformed cells developed slower, and none of the animals who received injections i.v. presented with metastases. BPV1-transformed cells had an intermediate tumorigenic and metastatic activity. Microvessels present in the different tumors were revealed by immunostaining with Griffonia (Bandeiraea) Simplicifolia lectin 1. Tumors obtained with c-Jun-transformed cells exhibited more neovascularization than those induced by the other oncogenes. By comparison to FR3T3 cells or SV40- or BPV1-transformed cells, c-Jun-transformed fibroblasts repress the antiangiogenic thrombospondin-1 and SPARC genes, whereas we found that they express higher levels of gene expression of the angiogenic vascular endothelial growth factor. Finally, as compared with cells before passage in animals, thrombospondin-1, SPARC, and VEGF gene expression was also deregulated in cell lines isolated from primary tumors induced by BPV1-transformants. Our results indicate that the high transforming potential of c-Jun, evidenced as soon as transformation is established in vitro, correlates with deregulation of gene expression of both angiogenic and antiangiogenic factors leading to rapid neovascularization of tumors.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

Kidney plays a major role in ammonia homeostasis after portasystemic shunting in patients with cirrhosis

The kidney plays an important role in ammonia metabolism. In this study the hypothesis was tested that the kidney can acutely diminish ammonia release after portacaval shunting. Thirteen patients with cirrhosis (6 female/7 male, age 54.4 +/- 3.3 yr) were studied. Blood was sampled prior to and 1 h after transjugular intrahepatic stent-shunt (TIPSS) insertion from the portal vein, a hepatic vein, the right renal vein, and the femoral vein, and renal and liver plasma flow were measured. Prior to TIPSS, renal ammonia release was significantly higher than ammonia release from the splanchnic region, which was not significantly different from zero. TIPSS insertion did not change arterial ammonia concentration or ammonia release from the splanchnic region but reduced renal ammonia release into the circulation (P < 0.05) to values that were not different from zero. TIPSS resulted in a tendency toward increased venous-arterial ammonia concentration differences across leg muscle. Post-TIPSS ammonia efflux via portasystemic shunts was estimated to be seven times higher than renal efflux. Kidneys have the ability to acutely diminish systemic ammonia release after portacaval shunting. Diminished renal ammonia release and enhanced muscle ammonia uptake are important mechanisms by which the cirrhotic patient maintains ammonia homeostasis after portasystemic shunting.     

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

Background

Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.

Results

Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al³⁺ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.

Conclusions

High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-153) contains supplementary material, which is available to authorized users.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.