A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive.

Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for phage replication. A single point mutation in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain. Upon infection of Escherichia coli by the temperature-sensitive phage at 42 degrees C, there is no detectable T7 DNA synthesis in vivo. DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 degrees C or 42 degrees C. Upon infection at 30 degrees C, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degrees C or 42 degrees C approach levels observed using wild-type T7 phage. The amount of soluble gene 5 protein produced at 42 degrees C is comparable to that produced at 30 degrees C, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 30 degrees C closely resemble that of wild-type T7 DNA polymerase. It is likely that the polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway.

Clinical information systems--developing a systematic planning process.

CHW is a large and diverse hospital system that developed a systematic planning process to define, prioritize, and assess the current status of those functionalities needed by caregivers to assist them in providing optimal care. In order to develop a durable consensus, a bottom-up approach was used. The details of the process including the structure of the meetings and many of the methodologies employed are presented.

Is computerization keeping up with patient-focused care?

It is essential to improve patient care in both quality and efficiency. Lessons gleaned from implementing bedside computing in a patient-focused care environment are presented. Although selective application of computer-based solutions can be effective, such efforts can also be risky and expensive.

Sensitivity, specificity, and risk of diagnostic procedures in a canine model of Streptococcus pneumoniae pneumonia.

A canine model in which unilateral Streptococcus pneumoniae pneumonia was induced was used to evaluate the sensitivity, specificity, and risk of various technics proposed for the diagnosis of pulmonary infection. Control and infected animals were studied during spontaneous and mechanical ventilation. We found that three procedures provided a reasonable degree of combined specificity and sensitivity: transthoracic needle aspiration (TTN), catheter-brush biopsy (CBB), and transbronchial biopsy (TBB). Transtracheal aspiration, although sensitive, was consistently low in specificity, whereas TTN provided the highest sensitivity/specificity. Both CBB and TBB provided almost the same results in intubated animals, but specificity was lower in nonintubated animals. Gram-stained smears obtained by TTN, CBB, and TBB were highly specific, but often negative. The only risk encountered was pneumothorax. The TTN was associated with a 20 to 30% risk of pneumothorax; CBB and TBB did not appear to induce pneumothorax. These studies provide the first animal data regarding the comparative yield/risk of these diagnostic approaches. As such, they may prove useful in the design of future human investigations.