Platelet transfusions in advanced hematological malignancies: a position paper.

Treatment of patients with advanced-stage hematological malignancies (HM) includes frequent transfusions. Given present limited hospital budgets, administrative pressure is increasing on hematology services to limit the cost of these transfusions. An expert multidisciplinary panel involved in hematology formed a working party to draw up a series of proposals, including definitions of advanced stage disease and the indications for platelet transfusion. Their proposals included: (a) Platelet transfusions are indicated for the treatment of bleeding caused by low platelet counts; (b) Patients should receive full information, including the basic criteria for platelet transfusion; (c) Doctors should be trained to assess whether or not platelet transfusions are urgently required; and (d) The practice of home transfusions should be encouraged.

Ribosomal protein L25 from Trypanosoma brucei: phylogeny and molecular co-evolution of an rRNA-binding protein and its rRNA binding site.

The gene encoding ribosomal protein L25, a primary rRNA-binding protein, was isolated from the protozoan parasite Trypanosoma brucei. Hybridization studies indicate that multiple copies of the gene are present per T. brucei haploid genome. The C-terminal domain of L25 protein from T. brucei is strikingly similar to L23a protein from rat, L25 proteins from fungal species, and L23 proteins from eubacteria, archaebacteria, and chloroplasts. A phylogenetic analysis of L23/25 proteins and the putative binding sites on their respective LSU-rRNAs (large subunit rRNAs) provides a rare opportunity to study molecular co-evolution between an RNA molecule and the protein that binds to it.

Blockage of AMV reverse transcriptase by antisense oligodeoxynucleotides.

Synthetic oligodeoxynucleotides, either unmodified or linked to an intercalating agent, have been used to prevent cDNA elongation by the AMV reverse transcriptase. Oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA part bound to the antisense oligonucleotide by the RNase-H activity associated with the retroviral polymerase.

Effect of RNA secondary structure and modified bases on the inhibition of trypanosomatid protein synthesis in cell free extracts by antisense oligodeoxynucleotides.

Every messenger RNA from leishmanias and trypanosomes has at its 5' end a conserved region termed the mini-exon sequence which, however, varies from species to species. In a systematic study mRNAs from Trypanosoma brucei, Trypanosoma vivax, and Leishmania enriettii were translated in cell-free extracts in the presence of oligodeoxynucleotides complementary to part of the mini-exon sequence. The affinity of the same oligonucleotides for target and non-target mRNAs was determined by thermal elution of filter-bound complexes showing that the critical temperature of half-dissociation of the complexes was linearly related to log (l + x), where l is the length of the oligomer and x its G + C content. A few oligomers exhibited a lower Tc value than expected which was ascribed to the presence of modified RNA bases or to the existence of a hairpin structure in the L. enriettii mini-exon. In most cases the efficiency of translation inhibition by the oligonucleotides was clearly correlated to their affinity for the target RNA. The modified bases weakened the inhibition of protein synthesis by oligonucleotides complementary to these regions.

[Antisense oligonucleotides: tools of molecular genetics and therapeutic agents]. / Les oligonucléotides antisens: outils de génétique moléculaire et agents thérapeutiques.

The binding of an oligodeoxynucleotide, so-called anti-sense, to the complementary sequence of a messenger RNA can prevent the synthesis of the encoded protein. This approach constitutes a very efficient and specific means to artificially regulate gene expression. Numerous chemical modifications have been introduced into synthetic oligos in order to provide them with properties that unmodified molecules do not display. For instance, oligos built up with methylphosphonate, phosphorothioate and alpha-anomer units lead to molecules that are resistant to DNases. Acridine-linked oligos exhibit an increased affinity for the target sequence due to the intercalation of the dye into the oligo/RNA duplex. Two different mechanisms account for translation inhibition by antisense oligos. Inhibition of the elongation step results only from the induced cleavage of the target RNA by RNase-H. In contrast, oligos targeted upstream of the AUG initiation codon can block the initiation step through an RNase-H independent mechanism. As a consequence, methylphosphonate- and alpha-oligos, which do not elicit RNase-H activity, targeted to the 5' region, are efficient antisense; but they are inactive if targeted to the coding sequence. Experiments performed with antisense oligos in cell-free extracts supported the notion that the mini-exon sequence, acquired by trans-splicing, was present on every message in trypanosomatids and on some of them in nematodes. Furthermore, an acridine-linked oligo complementary to the mini-exon sequence of Trypanosoma brucei induced a lethal effect on cultured procyclics. Therefore these compounds constitute promising tools in molecular genetics and could open new routes to rationally tailor therapeutic agents.

An acridine-linked oligodeoxynucleotide targeted to the common 5' end of trypanosome mRNAs kills cultured parasites.

Anti-messenger oligodeoxynucleotides covalently linked to an intercalating agent were tested for their ability to inhibit translation of Trypanosoma brucei mRNAs in a cell-free system. The sequence of these oligodeoxynucleotides was complementary to part of the 35-nucleotide (nt) sequence which is present at the 5' end of all trypanosome mRNAs (the so-called mini-exon sequence). In a rabbit reticulocyte lysate, a nonadeoxynucleotide linked to an acridine derivative, specifically inhibited protein synthesis from T. brucei mRNAs much more efficiently than unmodified oligodeoxynucleotides of similar length. These oligodeoxynucleotides were tested on cultured trypanosomes. The acridine-linked nonadeoxynucleotide had a lethal effect on the parasites. No effect was observed with the homologous unmodified 9-mer nor with those 9-mers linked to the acridine derivative which were not complementary to the mini-exon sequence. These effects are probably a result of hybrid formation between the anti-messenger and mini-exon sequence. Trypanocidal activity of the acridine-modified nonadeoxynucleotide is most likely due to (i) increased affinity for its target, (ii) improved resistance to 3' exonucleases, and (iii) promoted membrane penetration of living parasites.