TGIF1 is a negative regulator of MLL-rearranged acute myeloid leukemia.

Members of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing transcription factors are important downstream effectors of oncogenic fusion proteins involving the mixed lineage leukemia (MLL) gene. A well-characterized member of this protein family is MEIS1, which orchestrates a transcriptional program required for the maintenance of MLL-rearranged acute myeloid leukemia (AML). TGIF1/TGIF2 are relatively uncharacterized TALE transcription factors, which, in contrast to the remaining family, have been shown to act as transcriptional repressors. Given the general importance of this family in malignant hematopoiesis, we therefore tested the potential function of TGIF1 in the maintenance of MLL-rearranged AML. Gene expression analysis of MLL-rearranged patient blasts demonstrated reduced TGIF1 levels, and, in accordance, we find that forced expression of TGIF1 in MLL-AF9-transformed cells promoted differentiation and cell cycle exit in vitro, and delayed leukemic onset in vivo. Mechanistically, we show that TGIF1 interferes with a MEIS1-dependent transcriptional program by associating with MEIS1-bound regions in a competitive manner and that the MEIS1:TGIF1 ratio influence the clinical outcome. Collectively, these findings demonstrate that TALE family members can act both positively and negatively on transcriptional programs responsible for leukemic maintenance and provide novel insights into the regulatory gene expression circuitries in MLL-rearranged AML.

Irrigation with isoproterenol diminishes increases in pelvic pressure without side-effects during ureterorenoscopy: a randomized controlled study in a porcine model.

OBJECTIVE: Recently, we showed that endoluminally administered isoproterenol (ISO) inhibits muscle function of the pyeloureter in swine. This may be of value in managing increases in pelvic pressure during upper urinary tract endoscopy. The purpose of this study was to examine the effect of endoluminally administered ISO on increases in pelvic pressure and cardiovascular function during flexible ureterorenoscopy. MATERIAL AND METHODS: The study was performed in anaesthetized female pigs. In terms of endoscopic procedures, the pigs were randomized as follows: Group 1, irrigation with 0.1 microg/ml ISO added to saline (n=12); and Group 2, irrigation with saline (n=10). A 5-Fr catheter was retrogradely placed in the renal pelvis and an 8-Fr catheter in the bladder for pressure measurements. Flexible ureterorenoscopy was performed with constant irrigation at a perfusion rate of 8 ml/min. Pelvic, bladder and blood pressure and heart rate were registered continuously. RESULTS: Mean baseline pelvic pressure was identical in both groups: 12+/-2.3 mmHg in Group 1 and 14+/-3.6 mmHg in Group 2 (p=0.26). During ureterorenoscopy, mean pelvic pressure increased to 26+/-2.3 mmHg in Group 1 and to 38+/-3.1 mmHg in Group 2. Hence ISO reduced the pressure increase due to ureterorenoscopy by 42% (p<0.001). Pelvic pressure seemed to be independent of bladder pressure, which showed no difference between the two groups (p=0.067). Blood pressure and heart rate showed no significant differences between the two groups: p=0.425 and p=0.166, respectively. CONCLUSIONS: ISO (0.1 microg/ml) added to irrigation fluid significantly reduces the increase in pelvic pressure during ureterorenoscopy in pigs, without concomitant side-effects.

Local and systemic effects of endoluminal pelvic perfusion of isoproterenol: a dose response investigation in pigs.

PURPOSE: Isoproterenol (Sygehus Apotekerne Danmark, Copenhagen, Denmark) is a beta-adrenergic agonist known to cause upper urinary tract relaxation. We studied the local effect on pelvic pressure and the systemic effects of endoluminal perfusion with isoproterenol in a porcine model. MATERIALS AND METHODS: Pigs weighing 40 kg were studied. Catheters were placed in the renal pelvis for pressure measurement and perfusion, and a catheter was used to drain the bladder. Blood pressure and heart rate were recorded. In 6 pigs in group 1 the pelvic pressure increase was examined at increasing flow rates of 0, 2, 5, 8, 10 and 15 ml per minute with saline containing 0, 10(-3), 10(-2), 10(-1), 1 and 10 microg/ml isoproterenol. Blood values of isoproterenol were analyzed. In 6 pigs in group 2 the pelvis was perfused at a flow rate of 8 ml per minute with saline containing 0, 10(-5), 10(-4), 10(-3), 10(-2), 10(-1), 1 and 10 microg/ml isoproterenol. RESULTS: In group 1 endoluminal perfusion with isoproterenol inhibited the pelvic pressure increase due to perfusion at all concentrations of isoproterenol. At a perfusion rate of 8 ml per minute the maximal effect (a 78% decrease) was achieved using 0.1 microg/ml isoproterenol without cardiovascular side effects. In group 2 all isoproterenol concentrations caused significant inhibition of the pressure-flow relationship in a dose dependent matter. A 64% decrease in the pressure increase due to saline perfusion was achieved at 0.1 microg/ml isoproterenol without concomitant significant cardiovascular side effects. Isoproterenol was only detected in plasma during perfusion with 1 and 10 microg/ml isoproterenol, which caused significant cardiovascular side effects in the latter case. CONCLUSIONS: Isoproterenol significantly inhibits the pressure increase due to perfusion in the normal porcine renal pelvis without concomitant cardiovascular side effects. Isoproterenol is a safe drug in this porcine model and, hence, it is potentially useful during endourological procedures.

Host factor I, Hfq, binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability.

The stability of the ompA mRNA depends on the bacterial growth rate. The 5' untranslated region is the stability determinant of this transcript and the target of the endoribonuclease, RNase E, the key player of mRNA degradation. An RNA-binding protein with affinity for the 5' untranslated region ompA was purified and identified as Hfq, a host factor initially recognized for its function in phage Qbeta replication. The ompA RNA-binding activity parallels the amount of Hfq, which is elevated in bacteria cultured at slow growth rate, a condition leading to facilitated degradation of the ompA mRNA. In hfq mutant cells with a deficient Hfq gene product, the RNA-binding activity is missing, and analysis of the ompA mRNA showed that the growth-rate dependence of degradation is lost. Furthermore, the half-life of the ompA mRNA is prolonged in the mutant cells, irrespective of growth rate. Hfq has no affinity for the lpp transcript whose degradation, like that of bulk mRNA, is not affected by bacterial growth rate. Compatible with our results, we found that the intracellular concentration of RNase E and its associated degradosome components is independent of bacterial growth rate. Thus our results suggest a regulatory role for Hfq that specifically facilitates the ompA mRNA degradation in a growth rate-dependent manner.

The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly.

Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components.

Influence of Lewis alpha1-3/4-L-fucosyltransferase (FUT3) gene mutations on enzyme activity, erythrocyte phenotyping, and circulating tumor marker sialyl-Lewis a levels.

Fucosylated glycoproteins carrying alpha1-4 fucose residues are of importance for cell adhesion and as tumor markers. The Lewis gene, FUT3, encodes the only known alpha1-4-fucosyltransferase (FucT), and individuals who are deficient in this enzyme type as Lewis-negative on erythrocytes. We examined the mutational spectrum of the Lewis gene in Denmark and found 6 different mutations. Five, T59G, T202C, C314T, G508A, and T1067A, were frequent, and one, C445A, was only detected in one out of 40 individuals. Allele-specific polymerase chain reaction as well as cloning of FUT3 alleles showed that the 202 and 314 mutations were co-located on the same allele. COS7 cells transfected with an allele having the 202/314 mutations lacked enzyme activity. Polymerase chain reaction-cleavage assays were established for the genotyping of healthy individuals as well as 20 genuine Lewis-negative cancer patients and 10 non-genuine. The latter have Lewis-negative erythrocytes but saliva alpha1-4FucT activity. The genuine Lewis-negative individuals had mutations on both FUT3 alleles. In 66 healthy individuals, a gene dosage effect was detected as FUT3 heterozygous individuals had a lower alpha1-4FucT activity in saliva than did homozygous wild-type individuals. The lower enzyme level in heterozygous individuals resulted in a significantly (p < 0.04) lower level of circulating sialyl-Lewis a structure in serum. This has the clinical impact that cut-off levels in tumor marker assays should be defined on the basis of genotyping. In the group of non-genuine Lewis-negative cancer patients, whose erythrocytes convert from Lewis-positive to Lewis-negative during the disease, FUT3 heterozygosity was significantly (p < 0.05) more common.