Elongation factor 1beta is an actin-binding protein.

A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1beta. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium, indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1beta. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1beta. Direct interaction of eEF1beta with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1beta, recombinant preparations of Dictyostelium eEF1beta expressed in Escherichia coli, and the intact eEF1betagamma complex purified from wheat germ. Localization of eEF1beta in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1beta may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.

Interpretation of X chromosome dose at Sex-lethal requires non-E-box sites for the basic helix-loop-helix proteins SISB and daughterless.

For Drosophila melanogaster flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB (sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal (Sxl) in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless (da) proteins are known to form heterodimers that bind E-box sites and activate transcription. We examined SISB-Da binding at Sxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter Sxl(Pe). Surprisingly, most SISB-Da sites at Sxl(Pe) differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for Sxl(Pe) activity in embryos. Together with previous deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the Sxl(Pe) switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements.

The JAK/STAT signaling pathway is required for the initial choice of sexual identity in Drosophila melanogaster.

The choice of sexual identity in Drosophila is determined by a system that measures the X chromosome to autosome ratio (X/A). This system depends upon unequal expression of X-linked numerator genes in 1X and 2X nuclei. The numerators activate a special Sxl promoter, Sxl-Pe, in 2X/2A nuclei, but not 1X/2A nuclei. By multimerizing a conserved Sxl-Pe sequence block, we generated a gain-of-function promoter, Sxl-PeGOF, that is inappropriately active in 1X/2A nuclei. GOF activity requires the X-linked unpaired (upd) gene, which encodes a ligand for the Drosophila JAK/STAT signaling pathway. upd also functions as a numerator element in regulating wild-type Sxl-Pe reporters. We demonstrate that the JAK kinase, Hopscotch, and the STAT DNA-binding protein, Marelle, are also required for Sxl-Pe activation.

Survivors of catastrophic illness: outcome after direct transfer from intensive care to extended care facilities.

OBJECTIVE: To describe outcomes of adult survivors of prolonged critical illness after direct transfer to extended care facilities. DESIGN: A retrospective cohort study. SETTING: All adult intensive care units (ICUs) in a tertiary care university hospital. PATIENTS: A consecutive series of 97 adult survivors with an ICU stay of > or =7 days transferred directly from intensive care to extended care facilities between 1990 and 1996. INTERVENTIONS: None. METHODS AND MAIN RESULTS: Hospital and extended care facility charts were reviewed for patient characteristics, resource utilization, and survival. Survivors were for a minimum of 1 yr and a maximum of 6 yrs, and were interviewed to assess quality of life and functionality. The mean age of the patients was 66+/-16 (range, 19-93) yrs. The median length of ICU stay for these patients was 39 (range, 7-276) days. Only 18 of the 71 ventilator-assisted patients were weaned from mechanical ventilation after transfer to the extended care facility. Survival for the study period, at 1 yr after discharge from the ICU, was 49.5%. One year after discharge from the ICU, 11.5% of all patients had returned home, were breathing spontaneously, had a fair or better quality of life, and had good physical functionality. Each successive year, an increasing proportion of patients underwent direct transfer to an extended care facility. This strategy decreased the patients' length of stay (p<.002) in the ICU from year to year, but was significantly associated with an increase in readmissions to acute care hospitals (p<.002). CONCLUSIONS: Survivors of catastrophic illness who are so debilitated that they require transfer to an extended care facility have a low likelihood of achieving both survival and functional independence 1 yr after discharge from the ICU. Aggressive cost-conscious strategies to accelerate the transfer of these patients successfully reduced the length of ICU stay and hospital costs, but were associated with a high rate of readmission to tertiary care facilities.

Direct activation of Sex-lethal transcription by the Drosophila runt protein.

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. Here we demonstrate that runt also functions to activate transcription. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. Using an in vivo transient assay, we demonstrate that Runt's DNA-binding activity is essential for Sex-lethal activation in vivo. These experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. Moreover, we demonstrate that a small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo.

Rapid and complete fusion of macrophage lysosomes with phagosomes containing Salmonella typhimurium.

The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive after phagocytosis by macrophages. Although it is generally agreed that intracellular bacteria persist in membrane-bound phagosomes, there remains some question as to whether these phagosomes fuse with macrophage lysosomes. This report describes the maturation of phagosomes containing S. typhimurium inside mouse bone marrow-derived macrophages. Macrophages were infected briefly and incubated for various intervals; then they were examined by fluorescence microscopy for colocalization of bacteria with lysosomal markers. These markers included LAMP-1, cathepsin L, and fluorescent proteins or dextrans preloaded into lysosomes by endocytosis. By all measures, phagosomes containing S. typhimurium merged completely with the lysosomal compartment within 20 min of phagocytosis. The rate of phagosome-lysosome fusion was similar to the rate for phagocytosed latex beads. Phagolysosomes remained accessible to fluid-phase probes and contained lysosomal markers for many hours. Moreover, a large percentage of the wild-type bacteria that were viable 20 min after infection survived longer incubations inside macrophages, indicating that the survivors were not a minor subpopulation that avoided phagosome-lysosome fusion. Therefore, we conclude that S. typhimurium survives within the lysosomal compartments of macrophages.

A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm.

RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.

Nuf2, a spindle pole body-associated protein required for nuclear division in yeast.

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function.