Influencing sceptical staff to become supporters of service improvement: a qualitative study of doctors' and managers' views.

OBJECTIVE: To explore scepticism and resistance towards changes in working practice designed to achieve service improvement. Two principal questions were studied: (1). why some people are sceptical or resistant towards improvement programmes and (2). what influences them to change their minds. METHODS: Semi-structured qualitative interviews were conducted with 19 clinicians and 19 managers who held national and regional roles in two national programmes of service improvement within the NHS involving systematic organisational changes in working practices: the National Booking Programme and the Cancer Services Collaborative (now the Cancer Services Collaborative Improvement Partnership). RESULTS: Scepticism and resistance exist in all staff groups, especially among medical staff. Reasons include personal reluctance to change, misunderstanding of the aims of improvement programmes, and a dislike of the methods by which programmes have been promoted. Sceptical staff can be influenced to become involved in improvement, but this usually takes time. Newly won support may be fragile, requiring ongoing evidence of benefits to be maintained. CONCLUSIONS: The support of health service staff, particularly doctors, is crucial to the spread and sustainability of the modernisation agenda. Scepticism and resistance are seen to hamper progress. Leaders of improvement initiatives need to recognise the impact of scepticism and resistance, and to consider ways in which staff can become positively engaged in change.

Three forms of AU-1 like human rotaviruses differentiated by their overall genomic constellation and by the sequence of their VP8*.

Insight into the origin of human rotaviruses carrying the AU-1 VP4 allele was gained by examining their genomic RNA constellation using RNA-RNA hybridization and by sequencing the VP8* portion (nucleotides 1-750) of their gene 4. AU-1 like viruses isolated in Israel from children attending outpatient clinics were classified into three sub-genogroups based on RNA-RNA hybridization analysis: Subgenogroup 1 consists of two strains (Ro-5829 and Ro-5960) which belong to the AU-1 genogroup, since all their 11 segments hybridized to AU-1 segments. Subgenogroup 2 consists of one reassortant virus (Ro-5193) of which seven RNA segments hybridized to AU-1 segments and the remaining four segments hybridized to NCDV (bovine rotavirus). Subgenogroup 3 consists of four reassortant viruses (Ro-6460, Ro-6584, Ro-6784 and Ro-7044) which had a common genome constellation: only four of their RNA segments hybridized to AU-1 and the other seven segments hybridized to NCDV segments. Sequence analysis of the VP8* gene also revealed a three level pattern of homology with the AU-1 prototype and the local AU-1 like strains which was consistent with the overall genomic (RNA-RNA) constellation: Subgenogroup 1 had 98-98.1% homology with the AU-1 prototype; Subgenogroup 2 had 96.8% homology with the AU-1 prototype and 95.6-96.7% homology with Subgenogroup 1; Subgenogroup 3 had 95.3-95.6% homology with the prototype AU-1 and 93.4-94.3% homology with Subgenogroup 1. Possible evolutionary pathways are discussed.

Control of cell-type specific gene expression in Dictyostelium by the general transcription factor GBF.

To understand how positional information within an organism specifies patterning during development, we are analyzing spatially regulated gene expression in Dictyostelium. CAR3 is a member of the cAMP, 7-span receptor family which directs the transition from unicellular to multicellular organism and regulates cellular differentiation and pattern formation. CAR3 mRNA is expressed maximally at 8-10 hours of development, as individual cells aggregate and differentiate, and is accumulated to equivalent levels in all cells. CAR3 is also induced in shaking cultures by response to extracellular cAMP. We now show, by extensive mutagenesis, that the maximum length of contiguous sequences required for accurate spatiotemporal regulation of CAR3 is approx. 350 bp. These sequences include three significant elements located in upstream and transcribed regions. Arrays of G-boxes (GBF regulatory sites) are centered near positions -165 and +50 and, although either is sufficient for induction by cAMP and expression in prespore cells, both are required for expression in prestalk cells. Another GC-rich element near position -80 is required for maximal expression of prespore-specific constructs, although full-length promoters carrying clustered mutations through the -80 region are still expressed in all cells, but with slightly reduced expression. Spatiotemporal expression of CAR3 during development, thus, requires cell-specific combinatorial interactions of multiple but redundant regulatory components. These essential elements are located in upstream and transcribed regions. However, most surprisingly, a primary control for spatial patterning of CAR3 expression appears to be mediated by GBF, a general transcription factor expressed ubiquitously during Dictyostelium development following early aggregation.

The regulation of Dictyostelium development by transmembrane signalling.

Dictyostelium discoideum has a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughout Dictyostelium development to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type-specific gene expression.

Identification and targeted gene disruption of cAR3, a cAMP receptor subtype expressed during multicellular stages of Dictyostelium development.

Extracellular cAMP acts through cell-surface receptors to coordinate the developmental program of Dictyostelium. A cAMP receptor (cAR1), which is expressed during early aggregation, has been cloned and sequenced previously. We have identified a new receptor subtype, cAR3, that has approximately 56% and 69% amino acid identity with cAR1 and cAR2, respectively. cAR1, cAR2, or cAR3 expressed from plasmid in growing Dictyostelium cells can be photoaffinity labeled with 8-N3[32P]cAMP and phosphorylated when stimulated with cAMP. cAR3 RNA was not present during growth but appeared during late aggregation. Its expression peaked at 9 hr and then fell to a reduced level that was maintained until culmination. The expression of cAR3 protein followed a similar pattern, but with a 3-hr lag, and reached a maximum at the mound stage. In contrast, cAR1 protein was expressed predominantly during early aggregation and at low levels during later stages. At their respective peaks of expression, there were approximately 5 x 10(3) cAR3 sites per cell compared with approximately 7 x 10(4) cAR2 sites per cell. The cAR3 gene was disrupted by homologous recombination in several different parental cell lines. Surprisingly, the car3- cell lines display no obvious phenotype.

Protein U, a late-developmental spore coat protein of Myxococcus xanthus, is a secretory protein.

Protein U is a spore coat protein produced at the late stage of development of Myxococcus xanthus. This protein was isolated from developmental cells, and its amino-terminal sequence was determined. On the basis of this sequence, the gene for protein U (pru) was cloned and its DNA sequence was determined, revealing an open reading frame of 179 codons. The product from this open reading frame has a typical signal peptide of 25 amino acid residues at the amino terminal end, followed by protein U of 154 residues. This result indicates that protein U is produced as a secretory precursor, pro-protein U, which is then secreted across the membrane to assemble on the spore surface. This is in sharp contrast to protein S, a major spore coat protein produced early in development, which has no signal peptide, indicating that there are two distinct pathways for trafficking of spore coat proteins during the differentiation of M. xanthus.