G E Pugh | PubMed Health

PubMed1999

Interaction of the Escherichia coli DEAD box protein DbpA with 23 S ribosomal RNA.

The Escherichia coli DEAD box protein DbpA is unique among the DEAD box family in that its ATPase activity is specifically stimulated by bacterial 23 S ribosomal RNA. We have analysed the interaction between DbpA and a specific region within 23 S rRNA (namely nucleotides 2508-2580) which stimulates full ATPase activity. Using electrophoretic mobility shift assays we show that DbpA binds to this "specific" region with greater efficiency than to other regions of 23 S rRNA, and is not competed off by a non-specific RNA or a mutant RNA in which one of the stem-loops has been disrupted. These data suggest that the secondary structure within this region of 23 S rRNA is important for its recognition and binding by DbpA. We have also examined the ability of DbpA to unwind RNA and show that the purified protein does not behave as an RNA helicase in vitro with the substrates tested.

Adenosine Triphosphatases↗

PubMed1997

Distinct nuclear assembly pathways for lamins A and C lead to their increase during quiescence in Swiss 3T3 cells.

The expression of A-type lamins coincides with cell differentiation and as A-type lamins specifically interact with chromatin, a role in the regulation of differential gene expression has been suggested for A-type lamins. Using the mouse Swiss 3T3 cell line as a model, the change in two A-type lamins, lamins A and C, during cellular quiescence has been investigated. This well established model system mimics the first stages of differentiation when cells exit the cell cycle. In fact, quiescence in Swiss 3T3 cells was accompanied by a significant increase (2.6-fold) in lamin A protein levels and a smaller but reproducible increase (1.4-fold) in lamin C. These effects were fully reversible upon restimulation of the cells with serum. No effect upon lamin B levels was observed. Conversely, levels of A-type lamin mRNA decreased markedly as a result of quiescence suggesting transcriptional mechanisms are involved in the change in levels of lamins A and C. No difference in the incorporation of microinjected human lamin A into nuclei of quiescent or proliferating cells was observed. These data suggest A-type lamin binding sites were not limiting and indicated little difference between A-type lamin assembly mechanisms in quiescent and proliferating cells. The data did demonstrate lamin A and lamin C incorporation into the nuclear lamina proceeded by different pathways when microinjected in Swiss 3T3 cells. The incorporation of recombinant lamin C into the nuclear lamina was delayed compared to lamin A and proceeded via intranuclear foci. Such foci were not seen with microinjected lamin A. Instead, recombinant lamin A was rapidly (<20 minutes) incorporated into the nuclear lamina. Comicroinjection of lamin A with lamin C did not prevent foci formation but assisted in the rapid clearing (t1/2=30 minutes) of these structures and the incorporation of both lamins A and C into the lamina. These data suggest that the incorporation of lamin C into the lamina is facilitated by lamin A. They demonstrate a distinct difference in the nuclear assembly pathways of lamins A and C and show for the first time a functional distinction for these two splice variants of the A-type lamin gene. From the differences in assembly pathways and changes in protein levels accompanying quiescence in 3T3 cells, we suggest distinct roles for lamin A and lamin C in proliferating and quiescent states of the cell cycle.

3T3 Cells↗

PubMed1994

Computerized databases for emergency care: what impact on patient care?

A field-based evaluation is conducted of a Clinical Computerized Information System (CCIS). Following training, the use of the CCIS database, word processing and other programs by thirteen full-time practicing emergency physicians in two urban emergency departments of a University-associated teaching hospital was studied over a one-year period. A tracking program automatically logged frequency and duration of use by the physicians, and user satisfaction was assessed by a reliable and validated questionnaire instrument. Based on utilization data and verbal reports of these physicians, CCIS database searching was not only found to be easy-to-learn but was readily accessible during emergency shifts. Individual physicians were found to perform an average of 3.5 searches per month lasting a mean search time of 8 min. Positive notes about the CCIS system included ease-of-use, accuracy of data, accessibility of system, and value of output while negative perceptions included a lack of integration with other systems, a lack of system completeness, and a high subscription cost. It was suggested that a less costly telephone link to a high-volume Centre would be desirable in actual implementation of the system.

Attitude of Health Personnel↗