High-throughput production of Pyrococcus furiosus proteins: considerations for metalloproteins.

Free-living prokaryotic organisms contain all of the proteins required for the basic biochemical processes of life. As part of the Southeastern Collaboratory for Structural Genomics (SECSG), Pyrococcus furiosus is being used as a model system for developing a high-throughput protein expression and purification protocol. Its 1.9 million basepair genome encodes approximately 2200 putative proteins, less than 25% of which show similarity to any structurally characterized protein in the Protein Data Bank. The overall goal of the structural genomics initiative is to determine, in total, all existing protein folds. The immediate objective of this work is to obtain recombinant forms of all P. furiosus proteins in their functional states for structural determination. Proteins successfully produced by overexpression in another organism such as the bacterium Escherichia coli typically contain a single subunit, are soluble and do not contain (complex) cofactors. Analyses of the P. furiosus genome suggest that perhaps only a quarter of the genes encode proteins that would fall into this category. The hypothesis is that lack of the appropriate cofactor or of the partner protein(s) necessary to form a complex are major reasons why many recombinant proteins are insoluble. This work describes development of the production pipeline with attention to prediction and incorporation of cofactors.

Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants.

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) has recently been shown to participate in lignin biosynthesis in herbacious tobacco plants. Here, we demonstrate that CCoAOMT is essential in lignin biosynthesis in woody poplar (Populus tremula x Populus alba) plants. In poplar stems, CCoAOMT was found to be expressed in all lignifying cells including vessel elements and fibers as well as in xylem ray parenchyma cells. Repression of CCoAOMT expression by the antisense approach in transgenic poplar plants caused a significant decrease in total lignin content as detected by both Klason lignin assay and Fourier-transform infrared spectroscopy. The reduction in lignin content was the result of a decrease in both guaiacyl and syringyl lignins as determined by in-source pyrolysis mass spectrometry. Fourier-transform infrared spectroscopy indicated that the reduction in lignin content resulted in a less condensed and less cross-linked lignin structure in wood. Repression of CCoAOMT expression also led to coloration of wood and an elevation of wall-bound p-hydroxybenzoic acid. Taken together, these results indicate that CCoAOMT plays a dominant role in the methylation of the 3-hydroxyl group of caffeoyl CoA, and the CCoAOMT-mediated methylation reaction is essential to channel substrates for 5-methoxylation of hydroxycinnamates. They also suggest that antisense repression of CCoAOMT is an efficient means for genetic engineering of trees with low lignin content.

The utility of traditional prehospital interventions in maintaining thermostasis.

OBJECTIVE: Hypothermia can have a negative effect on the metabolic and hemostatic functions of patients with traumatic injuries. Multiple methods of rewarming are currently used in the prehospital arena, but little objective evidence for their effectiveness in this setting exists. The purpose of this study was to assess the relative effectiveness of traditional prehospital measures in maintaining thermostasis in trauma patients. METHODS: Participating helicopter and ground ambulance ALS units were prospectively randomized to provide either routine care only (passive or no warming) or routine care (passive warming) in conjunction with active warming (either reflective blankets, hot pack rewarming, or warmed IV fluids). A total of 174 trauma code patients, aged >14 years, who met inclusion criteria were prospectively enrolled by prehospital providers. Patients who received a non-assigned intervention or who had incomplete temperature data were dropped from the analysis. A total of 134 patients were included in the final analysis. RESULTS: Patients who received hot pack rewarming showed a mean increase in body temperature during transport (+1.36 degrees F/0.74 degrees C), while all other groups (no intervention, passive rewarming, reflective blankets, warmed IV fluids, warmed IV fluid plus reflective blanket) showed a mean decrease in temperature during transport [-0.34 to -0.61 degrees F (-0.2 to -0.4 degrees C); p<0.01]. In addition, the hot pack group was consistent, with every patient who received hot pack warming showing an increase in body temperature during transport, while in all other groups there were patients who had both increases and decreases in temperature. The intervention groups did not differ significantly on exposure to precipitation, transport unit temperature, total prehospital time, initial vital signs, amount of fluid administered, Injury Severity Score, or Glasgow Coma Score. CONCLUSIONS: Most traditional methods of maintaining trauma patient temperature during prehospital transport appear to be inadequate. Aggressive use of hot packs, a simple, inexpensive intervention to maintain thermostasis, deserves further study as a potential basic intervention for trauma patients.

Review of a computer based telephone Helpline in an A&E department.

OBJECTIVE: An audit of use of the telephone Helpline in the accident and emergency (A&E) department was conducted to establish the type of call, time, by whom the calls were made, appropriateness of advice given, and whether callers attended or not as advised, and also to obtain feedback from callers as to the degree of satisfaction with the advice given. METHODS: All calls over a four week period were included in the study. The information logged comprised name, age, telephone number, date and time of call, nature of query, advice given, and name of advisor. Callers whose telephone number had been noted were contacted for feedback. RESULTS: There were 300 calls over the four week period, and 150 callers were available for feedback. Most calls were received between 12.00 and 20.00 hours. The majority of callers were parent or self, 52% of calls were for problems concerning adults, and 40% for children; no age was recorded in 8%. Nursing staff answered 90.7% of the calls. Queries were wide ranging but commonly involved open and closed wounds and head injuries, with 29.3% being advised to attend A&E, 32.3% to ring or see their GP, and 38.4% were reassured or given other advice; 4% of callers attended A&E despite being advised otherwise, 97% were judged to have received appropriate advice, and 91.3% of callers were satisfied with the advice given. CONCLUSION: The Helpline has proved of benefit to the public. It is necessary and important to have protocols for common problems and to document all details carefully. It is also necessary that nursing staff are trained in handling the telephone queries and using the protocols. The use of a PC based system has improved data collection and also allows immediate access to protocols.