Improved solid-phase synthesis of alpha,alpha-dialkylated amino acid-rich peptides with antimicrobial activity.

A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid-phase synthetic techniques. Each nonapeptide was rich in alpha,alpha-dialkylated amino acids [one 4-aminopiperidine-4-carboxylic acid (Api) and six alpha-aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9-fluorenylmethyloxycarbonyl (Fmoc)-Aib-Aib-OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3(10)-helical, amphipathic design of these peptides was born out most prominently in the N-terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations < or =100 microM) and the acetylated peptides (concentrations < or = 200 microM) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.

Acute rhabdomyolysis after cardiac transplantation: a diagnostic conundrum.

A 13-year-old girl presented with right ventricular failure secondary to Ebstein's malformation (downward displacement of the tricuspid valve leaflets with adherence to the right ventricular muscle and redundancy or dysplasia of the tricuspid valve leaflets). She subsequently required a heart transplant but developed rhabdomyolysis early in the postoperative period and required ventilatory support for more than 3 weeks. A variety of causes were considered, but her condition improved only when cyclosporin was eliminated from the immunosuppression regimen. We believe it is likely that the rhabdomyolysis has been caused by cyclosporin. If so, this has occurred both earlier in the clinical course and at lower serum concentrations than previously described.

Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

Involvement of a tissue-specific RNA recognition motif protein in Drosophila spermatogenesis.

RNA binding proteins mediate posttranscriptional regulation of gene expression via their roles in nuclear and cytoplasmic mRNA metabolism. Many of the proteins involved in these processes have a common RNA binding domain, the RNA recognition motif (RRM). We have characterized the Testis-specific RRM protein gene (Tsr), which plays an important role in spermatogenesis in Drosophila melanogaster. Disruption of Tsr led to a dramatic reduction in male fertility due to the production of spermatids with abnormalities in mitochondrial morphogenesis. Tsr is located on the third chromosome at 87F, adjacent to the nuclear pre-mRNA binding protein gene Hrb87F. A 1.7-kb Tsr transcript was expressed exclusively in the male germ line. It encoded a protein containing two RRMs similar to those found in HRB87F as well as a unique C-terminal domain. TSR protein was located in the cytoplasm of spermatocytes and young spermatids but was absent from mature sperm. The cellular proteins expressed in premeiotic primary spermatocytes from Tsr mutant and wild-type males were assessed by two-dimensional gel electrophoresis. Lack of TSR resulted in the premature expression of a few proteins prior to meiosis; this was abolished by a transgenic copy of Tsr. These data demonstrate that TSR negatively regulated the expression of some testis proteins and, in combination with its expression pattern and subcellular localization, suggest that TSR regulates the stability or translatability of some mRNAs during spermatogenesis.

Primary structure of Drosophila ribosomal protein L14 and identification of conserved protein motifs.

Determination of the primary structure of individual ribosomal proteins is important for understanding their functions and organization within the ribosome. I have sequenced a cDNA that encodes a Drosophila homolog of the rat ribosomal protein L14. The cDNA sequence was 601 nucleotides long, with an open reading frame encoding a protein of 166 amino acids. Homology searches revealed 34-38% sequence identity to the rat and yeast L14 ribosomal proteins. There were also extensive homologies to sequences in the EST database, which are likely to encode portions of L14. Analysis of sequence comparisons revealed several highly conserved regions, one of which is related to a portion of ribosomal protein L27. The sizes of the L14 proteins vary between different species, with most of the variability confined to the C-terminal region.

Association of RB97D, an RRM protein required for male fertility, with a Y chromosome lampbrush loop in Drosophila spermatocytes.

The Y chromosome of Drosophila melanogaster, which is required only for male fertility, contains six loci that are essential for spermatogenesis. In primary spermatocytes, three of these loci form large lampbrush loops containing RNA transcripts and associated proteins. The identities and functions of these Y chromosome loop-binding proteins are largely unknown. This report demonstrates that the RB97D protein, which is essential for spermatogenesis, bound to a specific lampbrush loop. RB97D contains two copies of a well-characterized RNA binding domain, the RNA recognition motif, followed by a proline-glutamine rich domain. Immunohistochemical and immunofluorescence experiments showed that in the testis, RB97D was found only in primary spermatocyte nuclei and associated with the C loop from the ks-1 fertility locus in an RNAse-sensitive manner. The anti-RB97D antibodies also bound a single Y chromosome loop in D. hydei, suggesting that the protein and its loop-binding function have been evolutionarily conserved. These results demonstrate that the proteins that bind lampbrush loops can be essential for fertility. Since RB97D was present only premeiotically, its function is likely to be directly related to the metabolism of the C loop transcripts.

Altered levels of the Drosophila HRB87F/hrp36 hnRNP protein have limited effects on alternative splicing in vivo.

The Drosophila melanogaster genes Hrb87F and Hrb98DE encode the fly proteins HRB87F and HRB98DE (also known as hrp36 and hrp38, respectively) that are most similar in sequence and function to mammalian A/B-type hnRNP proteins. Using overexpression and deletion mutants of Hrb87F, we have tested the hypothesis that the ratio of A/B hnRNP proteins to SR family proteins modulates certain types of alternative splice-site selection. In flies in which HRB87F/hrp36 had been overexpressed 10- to 15-fold above normal levels, aberrant internal exon skipping was induced in at least one endogenous transcript, the dopa decarboxylase (Ddc) pre-mRNA, which previously had been shown to be similarly affected by excess HRB98DE/hrp38. In a second endogenous pre-mRNA, excess HRB87F/hrp36 had no effect on alternative 3' splice-site selection, as expected from mammalian hnRNP studies. Immunolocalization of the excess hnRNP protein showed that it localized correctly to the nucleus, specifically to sites on or near chromosomes, and that the peak of exon-skipping activity in Ddc RNA correlated with the peak of chromosomally associated hnRNP protein. The chromosomal association and level of the SR family of proteins were not significantly affected by the large increase in hnRNP proteins during this time period. Although these results are consistent with a possible role for hnRNP proteins in alternative splicing, the more interesting finding was the failure to detect significant adverse effects on flies with a greatly distorted ratio of hnRNPs to SR proteins. Electron microscopic visualization of the general population of active genes in flies overexpressing hnRNP proteins also indicated that the great majority of genes seemed normal in terms of cotranscriptional RNA processing events, although there were a few abnormalities consistent with rare exon-skipping events. Furthermore, in a Hrb87F null mutant, which is viable, the normal pattern of Ddc alternative splicing was observed, indicating that HRB87F/hrp36 is not required for Ddc splicing regulation. Thus, although splice-site selection can be affected in at least a few genes by gross overexpression of this hnRNP protein, the combined evidence suggests that if it plays a general role in alternative splicing in vivo, the role can be provided by other proteins with redundant functions, and the role is independent of its concentration relative to SR proteins.

The anaesthetic management of Caesarean section for placenta praevia: a questionnaire survey.

The purpose of this study was to determine current UK anaesthetic practice regarding the use of regional anaesthesia in the management of patients with placenta praevia presenting for Caesarean section. We asked the members of the Obstetric Anaesthetists Association to complete a postal questionnaire in which a range of clinical situations involving varying degrees of placenta praevia were presented. In each case respondents were asked whether they would be willing to use regional anaesthesia. A wide variety of clinical practice was demonstrated. Anaesthetists with two or more obstetric sessions were more willing to use regional anaesthesia for Caesarean section in the presence of placenta praevia in both elective and emergency situations associated with haemorrhage.

Posttranscriptional Regulation and RNA Binding Proteins in Development.

The precise spatial and temporal control of gene expression during the development of multicellular organisms is achieved by the use of both transcriptional and posttranscriptional control mechanisms. In fact, for some developmental processes, posttranscriptional regulation can be more important than transcriptional control. The mechanisms and proteins involved in posttranscriptional regulation are increasingly well understood. This review focuses on three well-characterized examples of posttranscriptional regulation in development, and highlights recent progress in each area. Copyright 1995 S. Karger AG, Basel

Cabeza, a Drosophila gene encoding a novel RNA binding protein, shares homology with EWS and TLS, two genes involved in human sarcoma formation.

We have previously described a partial Drosophila cDNA, clone P19, which bears homology to members of the RNA recognition motif (RRM) family of proteins [Haynes et al. (1987) Proc. Natl. Acad. Sci. USA, 84, 1819-1823]. RNA binding as well as involvement in RNA processing has been demonstrated for some RRM proteins. We report here the further characterization of P19, which we renamed cabeza (caz). caz is located on the X chromosome at position 14B. Using Northern analysis, at least four transcripts from the caz gene were observed at varying levels during development. caz mRNA and protein are enriched in the brain and central nervous system during embryogenesis. In addition, the protein is enriched in the adult head. UV crosslinking was used to demonstrate in vitro RNA binding activity for full-length recombinant caz protein and for the caz RRM domain. Sequence analysis revealed caz is related to two human genes, EWS and TLS, which are involved in chromosomal translocations. The fusion of EWS and TLS to other cellular genes results in sarcoma formation. In addition to their overall structural organization and sequence similarity, these three genes share an RRM which is divergent from typical RRMs. Therefore, it appears that these genes constitute a new sub-family of RNA binding proteins.

An assessment of the consistency of ASA physical status classification allocation.

The American Society of Anesthesiologists' (ASA) Physical Status Classification was tested for consistency of use by anaesthetists. A postal questionnaire was sent to 113 anaesthetists of varying experience working in the Northern Region of England. They were asked to allot ASA grades to 10 hypothetical patients. Ninety-seven (85.8%) responded to two mailings. In no case was there complete agreement on ASA grade, and in only one case were responses restricted to two of the five possible grades. In one case there was a significant difference in answers between anaesthetists with the FRCA (or equivalent) qualification, and those without. So much variation was observed between individual anaesthetist's assessments when describing common clinical problems that the ASA grade alone cannot be considered to satisfactorily describe the physical status of a patient.

The Rb97D gene encodes a potential RNA-binding protein required for spermatogenesis in Drosophila.

Many proteins that bind RNA contain a common RNA-binding domain, the RNP motif. We have been studying two Drosophila RNP motif proteins, Hrb98DE and Hrb87F, which are hnRNA-binding proteins. We report here the characterization of the Rb97D gene, which encodes a protein that is closely related to the Hrb proteins in the RNP motif domain, but has a distinctive proline-rich C-terminal domain. The gene is located at 97D on the right arm of the third chromosome, near the rough gene. Multiple transcripts from the Rb97D gene are present at varying levels throughout development. The transcripts are generated by alternative processing in the coding and 3' untranslated regions, and can encode two protein isoforms. Analysis of a mutant containing a P element inserted into the 5' untranslated region of the gene demonstrates that Rb97D is required for male fertility. Possible models for the function of Rb97D in testes are discussed.

Comparison of epidural methadone with epidural diamorphine for analgesia following caesarean section.

Analgesia provided by either 5 mg diamorphine, or 5 mg methadone administered by the epidural route during elective caesarean section was compared in 40 women. The median time to further analgesia in the methadone group was 395 min, and 720 min in the diamorphine group, P = 0.0003. Linear analogue scores to assess pain were measured 2-hourly for 12 h, then again at 24 h postoperatively. Pain scores were significantly lower in the diamorphine group at 8 and 10 h. The median cumulative i.m. morphine dose administered during the first 24 h was 20 mg in the methadone group and 0 mg in the diamorphine group (P = 0.0005). Nausea and pruritus were common side effects in both groups. Continuous pulse oximetry data were available for 12 h post-operatively in 15 patients receiving methadone, and in 17 patients receiving diamorphine. One or more episodes of significant desaturation (< 90% for 30 s), occurred in three patients receiving methadone, and in nine patients receiving diamorphine. Desaturation to 90-92% occurred in a further three patients given epidural diamorphine, and in one further patient given epidural methadone.

Accuracy of coagulation studies performed on blood samples obtained from arterial cannulae.

We have assessed the accuracy of coagulation studies in blood obtained from intra-arterial cannulae. Paired samples were studied in blood from 39 patients receiving intensive care; one sample was obtained by venepuncture and the other from an intra-arterial cannula after the apparatus deadspace plus 5 ml of blood had been discarded. Activated partial thromboplastin time (APTT) (with thromboplastin routinely used in our laboratory), prothrombin time (PT), thrombin time (TT), fibrinogen and heparin assays were measured on each sample. In 37 sample pairs, APTT was measured also using a different thromboplastin. The median difference between the sample pairs was 5.5 s for APTT (P = 0.032) and 1.0 s (P = 0.048) for TT, the times for arterial cannula samples being longer. There was no significant difference between arterial cannula and venepuncture samples for PT or fibrinogen concentration. Heparin assays revealed heparin contamination in samples obtained from arterial cannulae in 15 of 30 patients not receiving heparin. It is concluded that, when coagulation studies are performed using the techniques used routinely in our laboratory, a blood sample from an arterial cannula may give clinically misleading information because of contamination with small amounts of heparin, and that separate venepuncture is recommended.