Evaluation of zebrafish embryos as a model for assessing inhibition of hERG.

INTRODUCTION: It has been proposed that the analysis of heart rate in zebrafish embryos can be used to assess the potential effects of compounds on hERG. The purpose of this study was to investigate the effect of compounds on the heart rate and atrioventricular dissociation in zebrafish. The compounds investigated were chosen based on the association or lack of association with QTc prolongation in the clinic. METHODS: Three-day-old embryos were incubated in buffered embryo medium. On the day of the study, fish were placed in a petri dish containing 5.0 mL of embryo medium and 125 mg/L MS-222 anesthetic. Drugs to be tested were added to the medium from a stock solution to achieve the desired target concentration. Ten fish were incubated in each concentration of drug for 80 min. Beat rates of the atrium and ventricle were recorded after the incubation by counting beats of the respective chambers using standard brightfield stereomicroscopy. RESULTS: All of the compounds associated with QT prolongation induced dissociation between the atrium and ventricular rates except D,L-sotalol and procainamide. The concentrations that induced dissociation tended to be higher than the hERG IC50. None of the negative control compounds caused atrioventricular dissociation at clinically efficacious concentrations. DISCUSSION: In conclusion, the present data demonstrate that zebrafish can be utilized to assess the effects of chemicals on hERG. However, the practical use of this assay may be difficult because of the high concentrations that must be reached to see those pharmacological effects.

Effects of fasting on evaluation of gastrointestinal transit with charcoal meal.

INTRODUCTION: At the present time, most studies investigating gastrointestinal transit time with charcoal are conducted in fasted rats. It seems reasonable to hypothesize that the fasting state of rats could influence the effect a compound had on gastrointestinal transit time. The purpose of this study was to investigate the effects of food on the pharmacological effects on gastrointestinal transit. METHODS: For each drug investigated, two sets of 32 male Sprague-Dawley rats were used. One set was studied after being fasted for approximately 6 h, the second set was studied after free access to food. Each set had 4 groups of animals (n=8/group) that were administered different doses, allowing the assessment of the drug effect after fasting and after free access to food. Animals were administered 0, 10, 25, and 75 mg/kg of morphine; 0, 10, 20, and 40 mg/kg loperamide, or 0, 0.05, 0.5, and 3.0 mg/kg clonidine. At predetermined times, an activated charcoal suspension was administered by oral gavage. Thirty minutes after receiving the charcoal meal, rats were euthanized and the small intestine was removed. The length of the small intestine and the distance traveled by the charcoal were recorded. For each animal, gastrointestinal transit was calculated as the percentage of the distance traveled relative to the total length of the small intestine. RESULTS: Baseline (vehicle dosed animals) gastrointestinal transit was significantly greater in fasted versus fed rats. In fasted rats, morphine did not have a significant effect on transit. In fed rats, 25 and 75 mg/kg morphine caused a significant decrease in transit. In fed and fasted rats, 0.5 and 3 mg/kg clonidine caused a significant decrease in transit. Loperamide did not affect gastrointestinal transit in fed or fasted rats at doses up to 40 mg/kg. DISCUSSION: These data demonstrate that food does not reduce the sensitivity of the gastrointestinal transit time.

The synthetic peptide PFWT disrupts AF4-AF9 protein complexes and induces apoptosis in t(4;11) leukemia cells.

The MLL gene at chromosome band 11q23 is commonly involved in reciprocal translocations detected in acute leukemias. A number of experiments show that the resulting MLL fusion genes directly contribute to leukemogenesis. Among the many known MLL fusion partners, AF4 is relatively common, particularly in acute lymphoblastic leukemia in infants. The AF4 protein interacts with the product of another gene, AF9, which is also fused to MLL in acute leukemias. Based on mapping studies of the AF9-binding domain of AF4, we have developed a peptide, designated PFWT, which disrupts the AF4-AF9 interaction in vitro and in vivo. We provide evidence that this peptide is able to inhibit the proliferation of leukemia cells with t(4;11) chromosomal translocations expressing MLL-AF4 fusion genes. Further, we show that this inhibition is mediated through apoptosis. Importantly, the peptide does not affect the proliferative capacity of hematopoietic progenitor cells. Our findings indicate that the AF4-AF9 protein complex is a promising new target for leukemia therapy and that the PFWT peptide may serve as a lead compound for drug development.

MLL fusion partners AF4 and AF9 interact at subnuclear foci.

The MLL gene is involved in translocations associated with both acute lymphoblastic and acute myelogenous leukemia. These translocations fuse MLL with one of over 30 partner genes. Collectively, the MLL partner genes do not share a common structural motif or biochemical function. We have identified a protein interaction between the two most common MLL fusion partners AF4 and AF9. This interaction is restricted to discrete nuclear foci we have named 'AF4 bodies'. The AF4 body is non-nucleolar and is not coincident with any known nuclear structures we have examined. The AF4-AF9 interaction is maintained by the MLL-AF4 fusion protein, and expression of the MLL-AF4 fusion can alter the subnuclear localization of AF9. In view of other research indicating that other MLL fusion partners also interact with one another, these results suggest that MLL fusion partners may participate in a web of protein interactions with a common functional goal. The disruption of this web of interactions by fusion with MLL may be important to leukemogenesis.

The polycomb protein MPc3 interacts with AF9, an MLL fusion partner in t(9;11)(p22;q23) acute leukemias.

Polycomb group (PcG) proteins assemble to form large multiprotein complexes involved in gene silencing. Evidence suggests that PcG complexes are heterogeneous with respect to both protein composition and specific function. MPc3 is a recently described mouse Polycomb (Pc) protein that shares structural homology with at least two other Pc proteins, M33 and MPc2. All three Pc proteins bind another PcG protein, RING1, through a conserved carboxy-terminal C-box motif. Here, data are presented demonstrating that MPc3 also interacts with AF9, a transcriptional activator implicated in the development of acute leukemias. The carboxy-terminus of AF9 is fused to the MLL protein in leukemias characterized by t(9;11)(p22;q23) chromosomal translocations. Importantly, it is the carboxy-terminus of AF9 to which MPc3 binds. The AF9 binding site of MPc3 maps to a central, non-conserved, region of the polypeptide sequence. In contrast to MPc3, data indicate that the Pc protein M33 does not interact with AF9. This finding suggests a potentially unique role for MPc3 in linking a PcG silencing complex to a transcriptional activator protein.

Role of the 3' tRNA-like structure in tobacco mosaic virus minus-strand RNA synthesis by the viral RNA-dependent RNA polymerase In vitro.

A template-dependent RNA polymerase has been used to determine the sequence elements in the 3' untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3'-terminal 4 nucleotides of domain D1 indicated the importance of the 3'-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3' untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

Soluble, template-dependent extracts from Nicotiana benthamiana plants infected with potato virus X transcribe both plus- and minus-strand RNA templates.

We have developed a method to convert membrane-bound replication complexes isolated from Nicotiana benthamiana plants infected with potato virus X (PVX) to a soluble, template-dependent system for analysis of RNA synthesis. Analysis of RNA-dependent RNA polymerase activity in the membrane-bound, endogenous template extracts indicated three major products, which corresponded to double-stranded versions of PVX genomic RNA and the two predominant subgenomic RNAs. The endogenous templates were removed from the membrane-bound complex by treatment with BAL 31 nuclease in the presence of Nonidet P-40 (NP-40). Upon the addition of full-length plus- or minus- strand PVX transcripts, the corresponding-size products were detected. Synthesis was not observed when red clover necrotic mosaic dianthovirus (RCNMV) RNA 2 templates were added, indicating template specificity for PVX transcripts. Plus-strand PVX templates lacking the 3' terminal region were not copied, suggesting that elements in the 3' region were required for initiation of RNA synthesis. Extracts that supported RNA synthesis from endogenous templates could also be solublized using sodium taurodeoxycholate and then rendered template-dependent by BAL 31 nuclease/NP-40 treatment. The solubilized preparations copied both plus- and minus-strand PVX transcripts, but did not support synthesis from RCNMV RNA 2. These membrane-bound and soluble template-dependent systems will facilitate analyses of viral and host components required for PVX RNA synthesis.

Identification and analysis of a third mouse Polycomb gene, MPc3.

A new mouse Polycomb (Pc) gene, MPc3, has been identified. The MPc3 protein contains the highly conserved chromodomain and carboxy-terminal COOH box of other known Pc proteins from diverse species. Like other Pc proteins, MPc3 physically interacts with the RING finger proteins RING1A and dinG/RING1B. MPc3 maps to the distal arm of mouse chromosome 11 (11E2), a region that contains other known Pc genes in addition to several disease loci that may be linked to abnormal Pc gene function.

Restoration of a stem-loop structure required for potato virus X RNA accumulation indicates selection for a mismatch and a GNRA tetraloop.

The 5' region of potato virus X (PVX) RNA contains a stem-loop structure, stem-loop 1 (SL1), that is required for efficient plus-strand RNA accumulation. To determine how changes to individual elements in SL1 are accommodated by the virus, we inoculated PVX transcripts containing modifications in the terminal tetraloop (TL), stem C (SC), and stem D (SD) regions onto Nicotiana benthamiana plants and analyzed progeny RNAs over a series of passages. Several progeny RNAs isolated from plants inoculated with the TL mutants containing changes to the first nucleotide of the GAAA motif or deletion of the entire TL sequence were found to contain multiple A insertions within the terminal loop region. The wild-type TL motif, GAAA, was recovered for all TL mutants by the second passage, suggesting that the sequence and potential structure of this element are crucial for PVX infection. Revertant RNAs isolated from plants inoculated with mutants in SD and the central region of SC indicated that increased stem length is tolerated. Restoration of SD length to the 4 bp typical of the wild-type PVX RNA was accompanied by A insertion into loop C. Mutants with a conversion of the C55-C78 mismatch to a G-C pair, relocation of this mismatch within the central region of SC, or deletion of C55-C78 were unable to infect protoplasts and plants. In contrast, the mutant with a conversion of the C55-C78 mismatch to an A-C mismatch, which exhibited low levels of PVX plus-strand RNA in protoplasts, was able to infect plants and quickly reverted to the wild-type C-C mismatch. These data indicate that important sequence and secondary structural elements within SL1 are required for efficient viral infection and that multiple A insertions within the TL and loop C regions, potentially by polymerase stuttering, accompany restoration of SL1 structure.

Long-distance RNA-RNA interactions and conserved sequence elements affect potato virus X plus-strand RNA accumulation.

Conserved octanucleotide sequences located upstream of two major potato virus X (PVX) subgenomic RNAs (sgRNAs), as well as elements in the 5' end of the genome, affect accumulation of sgRNA. To determine if complementarity between these sequences is important for PVX RNA accumulation, we analyzed the effects of mutations within these elements and compensatory mutations in a tobacco protoplast system and in plants. Mutations in the 5' nontranslated region (NTR mutants) that reduced complementarity resulted in lower genomic RNA (gRNA) and sgRNA levels, whereas mutations to the octanucleotide elements affected only the corresponding sgRNA levels. However, for both the NTR and octanucleotide mutants, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Mutants containing changes in the NTR and compensatory changes in one of the octanucleotide elements restored levels of gRNA and the other sgRNA species with an unaltered octanucleotide element to those of wild-type. Although compensatory changes significantly increased levels of the sgRNA species with the modified octanucleotide element, levels were not restored to those of wild-type. Our data indicate that long distance RNA-RNA interactions and the sequences of the interacting elements are required for PVX plus-strand RNA accumulation.

Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae.

The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na+/Li+-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li+-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li+ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li+-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast.

Calcineurin. Structure, function, and inhibition.

Calcineurin is a serine-threonine specific Ca(2+)-calmodulin-activated protein phosphatase that is conserved from yeast to humans. Remarkably, this enzyme is the common target for two novel and structurally unrelated immunosuppressive antifungal drugs, cyclosporin A and FK506. Both drugs form complexes with abundant intracellular binding proteins, cyclosporin A with cyclophilin A and FK506 with FKBP 12, which bind to and inhibit calcineurin. The X-ray structure of an FKPB12-FK506-calcineurin AB ternary complex reveals that FKBP12-FK506 binds in a hydophobic groove between the calcineurin A catalytic and the regulatory B subunit, in accord with biochemical and genetic studies on inhibitor action. Calcineurin plays a key role in regulating the transcription factor NF-AT during T-cell activation, and in mediating responses of microorganisms to cation stress. These findings highlight the potential of yeast genetic studies to define novel drug targets and elucidate conserved elements of signal transduction cascades.

Stem-loop structure in the 5' region of potato virus X genome required for plus-strand RNA accumulation.

Computer-generated thermodynamic predictions and solution structure probing indicated two stem-loop structures, stem-loop 1 (SL1; nt 32-106) and stem-loop 2 (SL2; nt 143-183), within the 5' 230 nt of potato virus X (PVX) RNA. Because the existence of SL1 was further supported by covariation analysis of several PVX strains, the functional significance of this structure was investigated by site-directed mutational analysis in a tobacco protoplast system. In general, mutations that reduced genomic plus-strand RNA accumulation similarly affected coat protein accumulation, indicating that subgenomic plus-strand RNA was also affected. In contrast, minus-strand RNA levels remained relatively unchanged. Mutational analysis of the stem C (SC) region of SL1 indicated that pairing was more important than sequence, which was consistent with the covariation analysis. Alterations that increased length and stability of either SC or stem D (SD) were deleterious to plus-strand RNA accumulation. The formation of internal loop C between SC and SD, as well as specific nucleotides within this loop, were also required. Several modifications were made to the terminal GAAA tetraloop, a motif known for enhanced RNA stability. Both GANA and GAAG motifs resulted in wild-type levels of RNA accumulation. However, a UUCG tetraloop was detrimental, indicating that the sequence of this element was important beyond just providing stabilization of the structure. These data indicate that multiple features of SL1 are critical for accumulation of PVX plus-strand RNA.

History of coat protein-mediated protection.

A decade of research has proven that plants can be genetically engineered to resist virus infection through expression of viral CP genes, as well as other viral genes and sequences. Additional opportunities for development of resistant plants will require research focused on mechanisms of protection, improvements in expression vector design, and transformation of new crop species. As each of these technologies is utilized singly or in combination to generate resistant crop varieties, the full impact of such engineered resistance will be realized.

The Bmi-1 oncoprotein interacts with dinG and MPh2: the role of RING finger domains.

Experimentally-induced mutations in the C3HC4 RING finger domain of the Bmi-1 oncoprotein block its ability to induce lymphomas in mice. In this report, the role of the Bmi-1 RING finger in mediating protein-protein interactions is examined using the yeast two-hybrid system. Bmi-1 interacts directly with the RING finger protein dinG/RING1B. Heterodimerization of the two proteins requires the intact RING finger structures of both Bmi-1 and dinG. Although the RING finger domains are necessary for dimerization, they are not sufficient for this process as residues outside the C3HC4 motif are also required. Thus, binding specificity may be partly conferred by residues outside the RING motif. Both Bmi-1 and dinG interact with the Polyhomeotic protein MPh2 through binding domains apart from the RING finger. The data suggest a model whereby Bmi-1, dinG, and MPh2 form a stable heterotrimeric complex in which each protein contributes to the binding of the others.

Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation.

The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation of Nicotiana tabacum NT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.

The 5' nontranslated region of potato virus X RNA affects both genomic and subgenomic RNA synthesis.

A tobacco protoplast system was developed to analyze cis-acting sequences required for potato virus X (PVX) replication. Protoplasts inoculated with transcripts derived from a PVX cDNA clone or from clones containing mutations in their 5' nontranslated regions (NTRs) were assayed for RNA production by S1 nuclease protection assays. A time course of plus- and minus-strand-RNA accumulation indicated that both minus- and plus-strand PVX RNAs were detectable at 0.5 h postinoculation. Although minus-strand RNAs accumulated more rapidly than plus-strand RNAs, maximum levels of plus-strand RNAs were 40- to 80-fold higher. On the basis of these data, time points were chosen for determination of RNA levels in protoplasts inoculated with PVX clones containing deletions or an insertion in their 5' NTRs. Deletions of more than 12 nucleotides from the 5' end, internal deletions, and one insertion in the 5' NTR resulted in substantially decreased levels of plus-strand-RNA production. In contrast, all modified transcripts were functional for minus-strand-RNA synthesis, suggesting that elements in the 5' NTR were not essential for minus-strand-RNA synthesis. Further analysis of the 5' NTR deletion mutants indicated that all mutations that decreased genomic plus-strand-RNA synthesis also decreased synthesis of the two major subgenomic RNAs. These data indicate that cis-acting elements from different regions of the 5' NTR are required for plus-strand-RNA synthesis and that this process may be linked to synthesis of subgenomic RNAs.