Stable interference of EWS-FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target.

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS-FLI1 target genes still remain unknown and many have been identified in heterologous model systems. METHODS: We have developed a stable RNA interference model knocking down EWS-FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS-FLI1 and to identify genes that could contribute to tumourigenesis. RESULTS: EWS-FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS-FLI1 inhibition. We showed that TOPK is a new target gene of EWS-FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence. CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.

RT-PCR cloning, characterization and mRNA expression analysis of a cDNA encoding a type II asparagine synthetase in common bean.

Following a RT-PCR strategy based on the design of degenerate oligonucleotides resembling conserved domains of asparagine synthetase (AS; EC 6.3.5.4), we isolated a 2 kb cDNA clone (PVAS2) from root tissue of the common bean (Phaseolus vulgaris). PVAS2 encodes a protein of 584 amino acids with a predicted relative molecular mass of 65810 Da, an isoelectric point of 6.4, and a net charge of -7.2 at pH 7.0. The amino acid sequence of the protein encoded by PVAS2 is very similar to that encoded by the soybean SAS2 asparagine synthetase gene. The amino-terminal residues of the predicted PVAS2 protein are identical to the amino acids that constitute the glutamine-binding (GAT) domain of AS from other plant species, which suggests that the PVAS2 cDNA encodes a type II glutamine-dependent form of asparagine synthetase. Southern blot analysis indicates that the common bean AS is part of a small family composed of at least two genes. Expression analysis by Northern blot revealed that the PVAS2 transcript accumulates to a high level in roots and, to a lesser extent, in nodules and developing pods. Accumulation of the PVAS2 transcript in the root seems to be negatively regulated by light and sucrose, and positively regulated by nitrate.

[Presence of antibodies against Venezuelan equine encephalitis virus subtype VI in patients with acute febrile illness]. / Presencia de anticuerpos contra el virus de la encefalitis equina venezolana subtipo VI en pacientes con enfermedad aguda febril.

In Argentina, there is no record of human cases produced by Dengue virus (Flavivirus), but Paraguay and Brasil (neighbouring countries) have notified human outbreaks of Dengue Haemorrhagic Fever. In this report, we inform the serological results of a limited human outbreak of a Dengue-like acute illness that occurred in General Belgrano Island, Formosa, Argentina in April 1989. This island is 35 km far from Clorinda city of Paraguay river, with a human population of 150 inhabitants. The weather of this area is humid with abundant rainfall, favouring mosquitoes proliferation. Two samples of serum from 28 human notified cases were studied using hemagglutination inhibition test (HI), complement fixation (CF), and plaque reduction neutralization (NT) test in Vero cell cultures. All tested sera were negative to Dengue, St. Louis encephalitis, Yellow Fever, Bussuquara, Rocio, Eastern and Western Equine Encephalitis arboviruses as well as Influenza and Rubella viruses. By contrast, infection with Venezuelan equine encephalitis virus (VEE), subtype VI-AG80-663 strain was demonstrated (34.5% positive by HI, 39.1% by CF and 51.6% by NT). Seroconversion was detected by NT in six cases and only five were positive by CF. The 26.8% of the sera reacted also with VEE subtype I AB by NT. Considering that no cross reaction were detected in NT with these two subtypes, our results suggest that both viruses are concomitantly circulating in the studied area. Furthermore, the seroconversions detected with AG80-663 strain firmly indicate that during the outbreak this virus subtype was circulating in the island, although we could not assure that it was the causal agent of the acute disease.

Comparison of an antimicrobial adhesive drape and povidone-iodine preoperative skin preparation in dogs.

The antimicrobial efficacy of an adhesive drape applied after a 1-minute alcohol scrub was compared to a povidone-iodine (PI) skin preparation technique in dogs. Each technique was applied to both sides of 15 adult anesthetized dogs on premeasured, clipped areas of skin. Skin bacteria were quantified before, immediately after, and 1 hour after skin preparation. Predominant skin bacteria were isolated by swabbing the skin. The percentages of bacterial reduction immediately after and 1 hour after skin preparation, percentages of negative culture results, cultures with more than five colony-forming units, and the frequency of skin reactions were calculated and analyzed statistically. Drape adhesion was assessed subjectively. The percentage reduction in skin bacteria was significant for both techniques and comparable to that reported in humans. The adhesive drape was significantly less effective in both the immediate and 1-hour periods. Lift occurred in 66% of drape applications but was not associated with high bacterial counts. Acute contact dermatitis was more frequent after skin preparation with PI. There was no difference between the techniques in recovery of potential skin pathogens. The authors conclude that application of this antimicrobial adhesive drape after a 1-minute alcohol scrub is not as effective in the reduction of skin bacteria in dogs as is PI preparation of the skin.

Comparison of three skin preparation techniques in the dog. Part 1: Experimental trial.

Premeasured, clipped areas of skin on both sides of 30 adult dogs were prepared with povidone-iodine (PI), chlorhexidine gluconate (CG) with a saline rinse, or 4% CG with a 70% isopropyl alcohol rinse. Skin bacteria were quantified with Replicating Organism Detection and Counting (RODAC) plates and cultured for identification before, immediately after, and 1 hour after skin preparation. The percentages of bacterial reduction immediately and at hour 1 and the percentages of negative cultures, cultures with more than five colony-forming units (CFUs), and skin reactions were analyzed by analysis of variance and chi-square. The percentage of reduction in skin bacteria for all techniques was significant and comparable with that reported in humans. There were no significant differences between PI and CG results except that acute contact dermatitis was observed more frequently after skin preparation with PI. The authors conclude that for similar application times, PI and 4% CG rinsed with saline or 70% isopropyl alcohol are equally effective for up to 1 hour in the preoperative skin preparation of dogs.

Comparison of three skin preparation techniques. Part 2: Clinical trial in 100 dogs.

The skin of 100 dogs undergoing clean or clean-contaminated surgical procedures was prepared with povidone-iodine (PI) or 4% chlorhexidine gluconate (CG) with saline or 70% isopropyl alcohol rinse. Skin bacteria at the incision site were quantified with Replication Organism Detection and Counting (RODAC) plates immediately before and after skin preparation in the preparation room, in the operating room, and postoperatively. The percentage of bacterial reduction, negative cultures, cultures with more than five colony-forming units, and skin reactions for each technique were calculated for each sample period and analyzed with the analysis of variance and Fischer tests. The percentage of bacterial reduction for all techniques was significant and comparable with results of a previous experimental study. There were no significant differences in percentages of bacterial reduction between PI and the CG techniques for surgical times up to 8 hours. There were fewer negative cultures and more cultures with high bacterial counts with PI than with CG and saline after the cleansing scrub. There were fewer negative cultures after surgery with CG and alcohol than with the other two techniques. Duration of the surgical procedure did not significantly affect the culture results. Significantly more skin reactions occurred with PI. The authors conclude that PI and 4% CG with a saline rinse are equally effective in antimicrobial efficacy under clinical conditions. However, 4% CG with a 70% isopropyl alcohol rinse may be inferior in residual antimicrobial activity.

Cloning, characterization and mRNA expression analysis of PVAS1, a type I asparagine synthetase gene from Phaseolus vulgaris.

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris L.). A 2-kb cDNA clone of this gene (PVAS1) encodes a protein of 579 amino acids with a predicted molecular mass of 65,265 Da, an isoelectric point of 6.3, and a net charge of -9.3 at pH 7.0. The PVAS1 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS1 complemented an Escherichia coli asparagine auxotroph, which demonstrates that it encodes a glutamine-dependent AS. The PVAS1 protein showed the highest similarity to soybean SAS1, and piled up with other legume ASs to form an independent dendritic group of type-I AS enzymes. Northern blot analyses revealed that the expression pattern of PVAS1 resembles that of PVAS2, another AS previously described in the common bean. Unlike PVAS2, however, PVAS1 was not expressed in the nodule and was not repressed by light, suggesting different functions for these two AS genes.

Presencia de anticuerpos contra el virus de la encefalitis equina venezolana subtipo VI en pacientes con enfermedad aguda febril / [Presence of antibodies against Venezuelan equine encephalitis virus subtype VI in patients with acute febrile illness]

In Argentina, there is no record of human cases produced by Dengue virus (Flavivirus), but Paraguay and Brasil (neighbouring countries) have notified human outbreaks of Dengue Haemorrhagic Fever. In this report, we inform the serological results of a limited human outbreak of a Dengue-like acute illness that occurred in General Belgrano Island, Formosa, Argentina in April 1989. This island is 35 km far from Clorinda city of Paraguay river, with a human population of 150 inhabitants. The weather of this area is humid with abundant rainfall, favouring mosquitoes proliferation. Two samples of serum from 28 human notified cases were studied using hemagglutination inhibition test (HI), complement fixation (CF), and plaque reduction neutralization (NT) test in Vero cell cultures. All tested sera were negative to Dengue, St. Louis encephalitis, Yellow Fever, Bussuquara, Rocio, Eastern and Western Equine Encephalitis arboviruses as well as Influenza and Rubella viruses. By contrast, infection with Venezuelan equine encephalitis virus (VEE), subtype VI-AG80-663 strain was demonstrated (34.5

positive by HI, 39.1

by CF and 51.6

by NT). Seroconversion was detected by NT in six cases and only five were positive by CF. The 26.8

of the sera reacted also with VEE subtype I AB by NT. Considering that no cross reaction were detected in NT with these two subtypes, our results suggest that both viruses are concomitantly circulating in the studied area. Furthermore, the seroconversions detected with AG80-663 strain firmly indicate that during the outbreak this virus subtype was circulating in the island, although we could not assure that it was the causal agent of the acute disease.