The design, characteristics and predictors of mortality in the North of England Cellulitis Treatment Assessment (NECTA).

AIMS: Cellulitis is a common cause of acute medical admissions in UK hospitals. The factors that determine susceptibility to an acute admission or to mortality following hospital admission are poorly defined. METHODS: We studied a retrospective cohort of 568 patients with a diagnosis of cellulitis between 1 January 2001 and 31 December 2003 in the north-east of England to see whether we could determine these factors. We collected data on the factors that were associated with acute hospital admissions and survival. We used a primary end-point of deaths within 1 year of admission for cellulitis. RESULTS: The characteristics that identified patients at high risk of mortality were present in 39.9% of the cohort studied. The four most common of these characteristics were lower limb oedema 30.1% (95% CI: -26.0 to 34.1), ulceration 24% (95% CI: -20.2 to 27.8), previous myocardial infarction (MI) 19.9% (95% CI: -16.3 to 23.4) and blunt injury 18.7% (95% CI: -15.3 to 22.2). Significant predictors of mortality were: patient's age (p < 0.001), presence of penetrating injury (p < 0.001), previous MI (p < 0.001), presence of liver disease (p = 0.003), presence of lower limb oedema (p = 0.01) and long-term use of drugs that caused sodium and water retention (p < 0.001). Treatment with i.v. flucloxacillin was found to be a significant predictor of survival (odds ratio = 3.43, z =3.42. p < 0.001) at 360 days. CONCLUSION: Our results show that cellulitis as a cause of an acute medical admission may present with a variety of clinical features. Some of these clinical features can be used to predict mortality within 360 days of an acute hospital admission.

The role of exportin-t in selective nuclear export of mature tRNAs.

Exportin-t (Xpo-t) is a vertebrate nuclear export receptor for tRNAs that binds tRNA cooperatively with GTP-loaded Ran. Xpo-t antibodies are shown to efficiently block tRNA export from Xenopus oocyte nuclei suggesting that it is responsible for at least the majority of tRNA export in these cells. We examine the mechanism by which Xpo-t-RanGTP specifically exports mature tRNAs rather than other forms of nuclear RNA, including tRNA precursors. Chemical and enzymatic footprinting together with phosphate modification interference reveals an extensive interaction between the backbone of the TPsiC and acceptor arms of tRNAPhe and Xpo-t-RanGTP. Analysis of mutant or precursor tRNA forms demonstrates that, aside from these recognition elements, accurate 5' and 3' end-processing of tRNA affects Xpo-t-RanGTP interaction and nuclear export, while aminoacylation is not essential. Intron-containing, end-processed, pre-tRNAs can be bound by Xpo-t-RanGTP and are rapidly exported from the nucleus if Xpo-t is present in excess. These results suggest that at least two mechanisms are involved in discrimination of pre-tRNAs and mature tRNAs prior to nuclear export.

Identification of a nuclear export receptor for tRNA.

BACKGROUND: Transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear import and export receptors. The receptors identified to date are members of a family of Ran GTPase-binding proteins whose founding member is importin-beta. Interaction between these receptors and their cargo is regulated by the GTP-bound form of Ran. Export complexes form and import complexes disassemble on binding of RanGTP to the receptor. Yeast Los 1 p is a member of the importin-beta family with a poorly defined role in tRNA production. RESULTS: A human member of the importin-beta family that is distantly related to Los 1 p (21% identity) has been characterized. The protein shuttled between the nucleus and cytoplasm and interacts with tRNA in a RanGTP-dependent manner. Injection of the protein into the nuclei of Xenopus oocytes resulted in a specific stimulation of the export of tRNA from the nucleus and in relief of the competitive inhibition of tRNA export caused by the introduction of saturating amounts of nuclear tRNA. CONCLUSIONS: The human protein has the functional properties expected of a transport receptor that mediates export of tRNA from the nucleus. We therefore name the protein Exportin(tRNA).

Energy- and temperature-dependent in vitro export of RNA from synthetic nuclei.

We describe a novel assay for the study of RNA export from the nucleus in vitro. Nuclei are assembled in Xenopus egg extract on paramagnetic beads coated with DNA containing a specific template for transcription. T7 RNA polymerase, to which a nuclear localisation signal is attached, is added to the nuclei, and after its import into the assembled nuclei, transcription is allowed to proceed. The use of radioactive NTPs coupled with the possibility to purify the nuclei on a magnet and thus rapidly change the extract in which the nuclei are incubated allows pulse-chase labelling experiments. Using these protocols we show that U1 snRNA-derived templates are transcribed inside the synthetic nuclei, and that the transcripts leave the intact nuclei in a time-, temperature- and energy-dependent way. This offers the possibility of a biochemical approach to the dissection of RNA export.

A possible role for the guide RNA U-tail as a specificity determinant in formation of guide RNA-messenger RNA chimeras in mitochondrial extracts of Crithidia fasciculata.

Chimeric g(uide) RNA:pre-mRNA molecules are potential intermediates of the RNA editing process in kinetoplastid mitochondria. We have studied the characteristics of chimeric molecules formed in mitochondrial extracts of the insect trypanosomatid Crithidia fasciculata which had been supplied with synthetic NADH dehydrogenase (ND) subunit-7 gRNA and pre-mRNA variants. The ability of a gRNA to participate in chimera formation in this system depends on the possibility of base pairing with the pre-mRNA via the anchor sequence, but not on the presence of a U-tail or a full-length informational part. Chimeras formed with a specific gRNA:pre-mRNA pair displayed a large variation in length, due to variably sized 3' end truncations of the gRNA moieties and variation in the sites in the pre-mRNA to which the gRNAs were attached. Surprisingly, the presence of a U-tail in the gRNA for a large part determined the specificity of the linkage. In 60% of the cases gRNAs possessing a U-tail of at least one residue were attached to an editing site, whereas 75% of the gRNAs without Us were attached to non-editing sites. Furthermore, the chimera forming activity was greatly stimulated by the addition of ATP but not by AMP-CPP, an ATP-analogue with a non-hydrolyzable alpha-beta phosphate bond. This suggests the involvement in the chimera formation of an RNA ligase.

Novel pattern of editing regions in mitochondrial transcripts of the cryptobiid Trypanoplasma borreli.

In mitochondria of Kinetoplastida belonging to the suborder Trypanosomatina, the nucleotide sequence of transcripts is post-transcriptionally edited via insertion and deletion of uridylate residues. In order to shed more light on the evolutionary history of this process we have searched for editing in mitochondrial RNAs of Trypanoplasma borreli, an organism belonging to the suborder Bodonina. We have cloned and sequenced a 5.3 kb fragment derived from a 37 kb mitochondrial DNA molecule which does not appear to be a part of a network structure and have found genes encoding cytochrome c oxidase (cox) subunit 1, cox 2 and apocytochrome (cyt) b, and genes encoding the small and large subunit mitoribosomal RNAs. The order in which these genes occur is completely different from that of trypanosomatid maxicircle genes. The 5' and 3' termini of both the cytb and cox1 gene are cryptic, the protein coding sequences being created by extensive insertion/deletion of Us in the corresponding mRNA sections. Phylogenetic analyses of the protein and ribosomal RNA sequences demonstrated that the separation between T.borreli and Trypanosomatina was an early event, implying that U-insertion/deletion processes are ancient. Different patterns of editing have persisted in different lineages, however, since editing of cox1 RNA and of relatively small 3'-terminal RNA sections is not found in trypanosomatids. In contrast, cox2 RNA which is edited in trypanosomatids by the insertion of four Us, is unedited in T.borreli.

RNA editing in mitochondria of cultured trypanosomatids: translatable mRNAs for NADH-dehydrogenase subunits are missing.

RNA editing in mitochondria of kinetoplastid protozoa involves the posttranscriptional insertion and deletion of uridylate residues in protein encoding regions of pre-mRNAs. Editing is required to remove gene-encoded translational defects or to convert a nonsense sequence into a sense message. In cultured trypanosomatids, however, translationally defective pre-mRNAs for a number of NADH-dehydrogenase subunits are not converted into functional mRNAs by editing. In this report, the available data are discussed in the context of current models for RNA editing.

Implications of novel guide RNA features for the mechanism of RNA editing in Crithidia fasciculata.

We have determined the relative steady state concentration of the two Crithidia fasciculata guide (g)RNAs involved in editing the two domains of mRNAs for NADH dehydrogenase (ND) subunit 7. We found that, although there was an 8-fold difference between the molar ratio of these two gRNAs relative to the (pre)-mRNA, the two domains are edited with a very similar frequency (around 50%). Also, for the editing of a given domain, many gRNA species exist with the same 5' end but with a different 3' uridylation site. Approximately 20% of these short gRNAs do not contain the information required for editing a complete domain, which may explain the high incidence of partially edited RNAs. Remarkably, genomically encoded Us are missing from two sites of a few of the gRNAs involved in editing apocytochrome b RNA. We speculate that these species are created by editing-like events. Both the short and complete forms of the ND7 gRNAs are found in chimeric molecules, in which the gRNA is covalently linked via its 3'-terminus to an editing site of pre-edited ND7 RNA. Some features of the chimeric molecules are at odds with current models of RNA editing: (i) U residues are completely absent from the connecting sequence of a number of these molecules, (ii) the ND7 gRNAs are frequently hooked up to the wrong editing domain of ND7 RNA, although other gRNAs are not found at these positions and (iii) in some chimeric molecules the gRNA appears to be linked to the 5' end of pre-edited RNA.

Conserved genes encode guide RNAs in mitochondria of Crithidia fasciculata.

RNA editing is the post-transcriptional alteration of the nucleotide sequence of RNA, which in trypanosome mitochondria is characterized by the insertion and deletion of uridine residues. It has recently been proposed that the information for the sequence alteration in Leishmania tarentolae is provided by small guide (g) RNAs encoded in the mitochondrial DNA [Blum et al. (1990) Cell, 60, 189-198]. We are studying the mechanism of RNA editing in the insect trypanosome Crithidia fasciculata and report that: (i) a full length, conventional DNA gene or an independently replicating RNA gene that could encode the edited MURF3 transcript is absent when probed for in sensitive, calibrated assay systems; (ii) in all cases (seven) investigated in C. fasciculata so far, putative gRNA genes are found in a position in the mitochondrial DNA virtually identical to that in L. tarentolae and (iii) also in C. fasciculata, the putative gRNA genes are transcribed into small RNAs with discrete 5' ends. These results provide strong evolutionary evidence in support of the participation of gRNAs in RNA editing. Remarkably, in C. fasciculata the basepaired region of some putative gRNA:mRNA hybrids contains a C:A non-Watson-Crick basepair.

RNA editing in transcripts of the mitochondrial genes of the insect trypanosome Crithidia fasciculata.

With the aid of cDNA and RNA sequence analysis, we have determined to what extent transcripts of mitochondrial maxicircle genes of the insect trypanosome Crithidia fasciculata are altered by RNA editing, a novel mechanism of gene expression which operates via the insertion and deletion of uridine residues. Editing of cytochrome c oxidase (cox) subunit II and III transcripts and of maxicircle unidentified reading frame (MURF) 2 RNA is limited to a small section and results in the creation of a potential AUG translational initiation codon (coxIII, MURF2) or the removal of a frameshift (coxII). No differences with the genomic sequences were observed in the remainder of these RNAs. Surprisingly, NADH dehydrogenase subunit I transcripts were completely unedited in the coding region, implying that an AUG translational initiation codon is absent. The partial ribosomal RNA sequences determined also conform to the gene sequences. Together these results lead to the conclusion that the unusual sequences predicted by the protein and rRNA genes must indeed be present in the gene products. Editing also occurred in the poly(A) tail of RNAs from all protein genes, including those that are unedited in the coding region. The tails display a large variation in AU sequence motifs. Finally, some cDNAs contained sequences absent from both the DNA and the edited RNA. Some of these may represent intermediates in the RNA editing process. We argue, however, that long runs of T may be artefacts of cDNA synthesis.