The effects of microchipping C57BL/6N mice on standard phenotyping tests.

The C57BL/6N inbred lines of mice are widely used in genetic research. They are particularly favoured in large scale studies such as the International Mouse Phenotyping Consortium (IMPC), where C57BL/6N mice are genetically altered to generate a collection of null alleles (currently more than 8500 null alleles have been generated). In this project, mice carrying null alleles are subjected to a pipeline of broad-based phenotyping tests to produce wide ranging phenotyping data on each model. We have previously described the development of a Home Cage Analysis system that automatically tracks the activity of group housed mice from a microchip inserted in the groin. This platform allows assessment of multiple biologically relevant phenotypes over long periods of time without experimenter interference, and therefore is particularly suited for high through-put studies. To investigate the impact of microchips on other tests carried out in the IMPC pipeline, we inserted microchips in 12 male and 12 female C57BL/6Ntac mice at seven weeks of age. Starting at nine weeks of age these mice underwent standard phenotyping tests, concurrently with 20 unchipped C57BL/6Ntac mice (10 females, 10 males). Tissues from a subset of the microchipped mice (six males and six females), chosen at random, were also sent for histopathological examination at the end of the phenotyping pipeline. No significant impact of insertion of microchip was observed in any of the phenotyping tests apart from bone mineral density measurement at DEXA due to the nature of the microchip. We therefore recommend that the microchip be inserted during the DEXA procedure, after the measurement is taken but before the mouse has recovered from the anaesthetic. This would avoid multiple anaesthetic exposures and prevent the potential variability in DEXA analysis output.

Managers see the problems associated with coding clinical data as a technical issue whilst clinicians also see cultural barriers.

OBJECTIVE: In UK general practice, the coding of clinical data (Read Coding) is far from universal. This study set out to examine the barriers to recording structured information in computerised medical records; and to explore whether managers and clinicians had different perspectives in how these barriers should be overcome. METHOD: A qualitative study, using semi-structured interviews of general practitioners, primary care nurses and practice managers. The interviews were recorded verbatim, and then underwent thematic analysis; additional interviews were conducted until thematic saturation was achieved. RESULTS: For clinicians the recording of structured data within a consultation is not a neutral activity, they are highly aware of diagnostic uncertainty and sensitive to the potential impact of both a correct and incorrect diagnostic label on their relationship with their patient. Clinicians accept that data has to be coded if they are to demonstrate that appropriate evidence based care has been provided to populations; but alongside this they require free-text as a more powerful reminder of the individual human encounter. Managers felt that they could encourage clinicians to code data for re-use as part of population data or as quality target indicators rather than as an enabler of the next consultation. CONCLUSIONS: The primary care consultation is a complex social interaction, and coding of the medical diagnosis in itself imposes the bio-medical model, carries assumptions about certainty, and is perceived by clinicians to potentially jeopardise their relationships with their patient. Further research to elicit patients' views may help clarify the magnitude of this barrier.

Development of an assessment tool to measure the influence of clinical software on the delivery of high quality consultations. A study comparing two computerized medical record systems in a nurse run heart clinic in a general practice setting.

A rating scale was developed to assess the contribution made by computer software towards the delivery of a quality consultation, with the purpose of informing the development of the next generation of systems. Two software programmes were compared, using this scale to test their ability to enable or inhibit the delivery of an ideal consultation with a patient with heart disease. The context was a general practice based, nurse run clinic for the secondary prevention of heart disease. One of the programmes was customized for this purpose; the other was a standard general practice programme. Consultations were video-recorded, and then assessed by an expert panel using the new assessment tool. Both software programmes were oriented towards the implementation of the evidence, rather than facilitating patient-centred practice. The rating scale showed, not surprisingly, significantly greater support from the customized software in the consultation in five out of eight areas. However, the scale's reliability measured by Cronbach's Alpha, was sub-optimal. With further refinement, this rating scale may become a useful tool that will inform software developers of the effectiveness of their programmes in the consultation, and suggest where they need development.

Circularization of mRNA by eukaryotic translation initiation factors.

Communication between the 5' cap structure and 3' poly(A) tail of eukaryotic mRNA results in the synergistic enhancement of translation. The cap and poly(A) tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S. cerevisiae through their interactions with different parts of the translation factor eIF4G. Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA. Our results suggest that formation of circular mRNA by translation factors could contribute to the control of mRNA expression in the eukaryotic cell.

Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation.

The yeast translation factor eIF4G associates with both the cap-binding protein eIF4E and the poly(A)-binding protein Pab1p. Here we report that the two yeast eIF4G homologs, Tif4631p and Tif4632p, share a conserved Pab1p-binding site. This site is required for Pab1p and poly(A) tails to stimulate the in vitro translation of uncapped polyadenylylated mRNA, and the region encompassing it is required for the cap and the poly(A) tail to synergistically stimulate translation. This region on Tif4631p becomes essential for cell growth when the eIF4E binding site on Tif4631p is mutated. Pab1p mutations also show synthetic lethal interactions with eIF4E mutations. These data suggest that eIF4G mediates poly(A) tail stimulated translation in vitro, and that Pab1p and the domain encompassing the Pab1p-binding site on eIF4G can compensate for partial loss of eIF4E function in vivo.

Computer generated slides--cheap!

Thirty-five millimeter slides can enhance the quality of presentations. They can present complex ideas and transmit information more clearly. They can engage the learner and add a touch of professionalism. However, the cost of professionally generated slides may be prohibitive for individuals working for smaller institutions with limited budgets. This article describes a method that can be used to produce high quality color slides with a fast turnaround time at a reasonable cost. Equipment needed is a computer with a graphics program and a suitably equipped camera.

Effects of growth hormone secretagogues in the transgenic growth-retarded (Tgr) rat.

Exogenous GH inhibits endogenous GH release by hypothalamic feedback. We have recently exploited this to generate transgenic growth-retarded (Tgr) rats, in which human GH is expressed in the hypothalamus, under the control of the rat GRF gene promoter. These rats show reduced pituitary size, GH deficiency, and dominant dwarfism, but are large enough for serial blood sampling studies to examine their spontaneous GH secretion and responses to GRF, somatostatin, and GH-releasing peptide-6 (GHRP-6). Like their normal wild-type littermates, Tgr rats show a sexually dimorphic pattern of GH secretion; males secrete GH in 3-h episodes, whereas females exhibit a more continuous irregular output, with higher baseline GH levels. In anesthetized male Tgr rats, the GH responses to GRF or GHRP-6 were markedly reduced compared with those of their nontransgenic littermates, but the differences were smaller in females. Despite the reduction in pituitary GH, peak plasma GH responses to serial GRF injections in conscious Tgr males or intermittent somatostatin infusions in conscious Tgr females were indistinguishable from the responses in their wild-type littermates. Furthermore, 7-day iv infusions of GRF (12.5-100 micrograms/day), given either continuously or as a pulsatile infusion stimulated growth in Tgr rats, as did pulsatile infusions of GHRP-6. Thus, despite their pituitary GH deficiency and dwarfism, Tgr rats maintain a sexually dimorphic pattern of GH release and can produce large GH secretory responses to exogenous secretagogues. They represent the first genetic model of GH deficiency in the rat in which dwarfism can be corrected by treatment with exogenous GH secretagogues.

Dominant dwarfism in transgenic rats by targeting human growth hormone (GH) expression to hypothalamic GH-releasing factor neurons.

Expression of human growth hormone (hGH) was targeted to growth hormone-releasing (GRF) neurons in the hypothalamus of transgenic rats. This induced dominant dwarfism by local feedback inhibition of GRF. One line, bearing a single copy of a GRF-hGH transgene, has been characterized in detail, and has been termed Tgr (for Transgenic growth-retarded). hGH was detected by immunocytochemistry in the brain, restricted to the median eminence of the hypothalamus. Low levels were also detected in the anterior pituitary gland by radioimmunoassay. Transgene expression in these sites was confirmed by RT-PCR. Tgr rats had reduced hypothalamic GRF and mRNA, in contrast to the increased GRF expression which accompanies GH deficiency in other dwarf rats. Endogenous GH mRNA, GH content, pituitary size and somatotroph cell number were also reduced significantly in Tgr rats. Pituitary adrenocorticotrophic hormone (ACTH) and thyroid-stimulating hormone (TSH) levels were normal, but prolactin content, mRNA levels and lactotroph cell numbers were also slightly reduced, probably due to feedback inhibition of prolactin by the lactogenic properties of the hGH transgene. This is the first dominant dwarf rat strain to be reported and will provide a valuable model for evaluating the effects of transgene expression on endogenous GH secretion, as well as the use of GH secretagogues for the treatment of dwarfism.

CUS1, a suppressor of cold-sensitive U2 snRNA mutations, is a novel yeast splicing factor homologous to human SAP 145.

The function of U2 snRNA in splicing is mediated by the proteins of the U2 small nuclear ribonucleoprotein. To identify proteins that influence the function of U2 snRNA we carried out a screen for mutations in Saccharomyces cerevisiae that suppress the cold-sensitive growth defect of a mutation in U2 stem loop IIa, a structure important for the stable association of the U2 snRNP with pre-mRNA. The screen identified three dominant suppressor genes, one of which, CUS1-54, encodes an essential splicing protein required for U2 snRNP addition to the spliceosome. The suppressor protein rescues the spliceosome assembly defect of the mutant U2 in vitro, indicating that suppression is direct. Allele specificity tests show that the suppressor does not simply bypass the requirement for U2 stem loop IIa. Extra copies of wild-type CUS1, but not CUS1-54, suppress the temperature-sensitive prp11 and prp5 mutations, linking CUS1 protein to a subset of other factors required at the same step of spliceosome assembly. CUS1 is homologous to SAP 145, a component of the mammalian U2 snRNP that interacts with pre-mRNA. The yeast genome also encodes a homolog of human SAP 49, a protein that interacts strongly with both SAP 145 and pre-mRNA, underscoring the conservation of U2 snRNP proteins that function in spliceosome assembly.

Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

Neuropathological distinction between Parkinson's dementia and Parkinson's plus Alzheimer's disease.

The distinctive clinical features of dementia in Parkinson's disease (PDD) and Parkinson's plus Alzheimer's disease (PD + AD) suggest different patterns of cerebral atrophy in these conditions. To determine the pathoanatomical substrates of dementia in PDD and PD + AD, morphometric analysis of 5 standardized coronal slices was used to identify volumetric changes in cerebral tissue. In PDD (n = 4) there were 9 to 23% reductions in cross-sectional area of cerebral cortex, a 38% loss of tissue in the globus pallidus + putamen, and an 18% reduction in area of the amygdala, whereas in PD + AD (n = 6) there was severe global atrophy of the cerebral cortex (27-29% reductions), moderate atrophy of white matter (10-19% reductions), and 40% reductions in areas of globus pallidus + putamen and the amygdala relative to neuropathologically intact controls (n = 14). Immunostaining with anti-glial fibrillary acidic protein disclosed significant gliosis of all four major subdivisions of neocortex in PD + AD and gray matter of the caudate, putamen, globus pallidus, and thalamus in both PDD and PD + AD relative to controls. The findings suggest that dementia in PDD is mainly subcortical in origin and due to neuronal degeneration in basal ganglia, the amygdala, and thalamus. In PD + AD the same pattern and degree of subcortical degeneration is evident, but there are clearly superimposed lesions involving cortical neurons and long projection fibers coursing through cerebral white matter that most likely account for the distinctive manifestations of dementia in this condition compared with PDD.

Tryptophan auxotrophs of Rhizobium japonicum.

Eleven tryptophan-requiring mutants of Rhizobium japonicum I-110 ARS were isolated after nitrous acid mutagenesis and fell into five groups based on characterization by supplementation with intermediates and enzyme assays.