BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip

While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system. We show here that the histidine (His) tag, routinely used in protein purification and in detection is an ideal tag for immobilization, despite the intrinsically low affinity between an immobilized metal ion and the His tag. This is due to strong rebinding effects caused by the high surface density of immobilized Ni2+-nitrilotriacetic acid (NTA) on the chips used here. The immobilization of the ligand can be adjusted to a low level using the same chip, such that mass transport limitation and rebinding of the analyte to the immobilized ligand is minimal. Nine different proteins with different numbers of His tags were tested for stable binding to the Ni2+-NTA surface. Most proteins with one His tag dissociate very rapidly from the Ni2+-NTA surface, and the KD for the interaction between His tag and Ni2+-NTA was estimated to about 10(-6) m at neutral pH. In contrast, two His tags are usually found to be sufficient for stable binding. The kinetics of the chaperonin system of Escherichia coli GroEL and GroES were analyzed as a model using this system and found to be very similar to those obtained with covalently immobilized ligands. The sensor chip can be reused many times, because of the powerful regeneration methods. The ligand can be freshly immobilized after each cycle, thus eliminating potential denaturation upon regeneration as a source of error.     

EBIC-guidelines for management of severe head injury in adults. European Brain Injury Consortium

Guidelines for the management of severe head injury in adults as evolved by the European Brain Injury Consortium are presented and discussed. The importance of preventing and treating secondary insults is emphasized and the principles on which treatment is based are reviewed. Guidelines presented are of a pragmatic nature, based on consensus and expert opinion, covering the treatment from accident site to intensive care unit. Specific aspects pertaining to the conduct of clinical trials in head injury are highlighted. The adopted approach is further discussed in relation to other approaches to the development of guidelines, such as evidence based analysis.     

Monosialoganglioside (GM1) immunofluorescence in rat spinal roots studied with a monoclonal antibody

The rate of reaction of monochlorobimane with glutathione (GSH) was measured in native human mammary MCF-7 adenocarcinoma cells (MCF-7wt) and sublines displaying resistance to 4-hydroperoxycyclophosphamide (MCF-7hc) and adriamycin (MCF-7adr) prior to examination by epifluorescence and confocal microscopy. After a 60-min incubation period at 37 degrees C, essentially all GSH was conjugated in the MCF-7wt and MCF-7adr cell lines whereas only 80% of the GSH was conjugated in the MCF-7hc line. All three lines displayed significant export of the conjugate from the cell during this period, with the MCF-7adr line displaying the most rapid efflux with 85% of the conjugate exported within 60 min. Epifluorescence microscopy detected an approximately 20% increase in integrated fluorescence intensity in the nuclear region in all three lines. Confocal microscopy however, indicated that most of the cells examined showed a homogeneous fluorescence distribution. The cells grown in monolayers were found to be thicker in the nuclear region suggesting that the observed increase in fluorescence intensity in the nuclear region in the images from epifluorescence microscopy was probably derived from fluorescence from an out-of-focus plane. Cells depleted of GSH with buthionine sulfoximine followed by treatment with mBCl showed significant fluorescence intensity resulting from nonspecific binding of this probe. These studies illustrate the need for measuring the rate of GSH conjugate export and for determining probe specificity, and emphasizes the need for using confocal techniques for the quantitative evaluation of the distribution of intracellular fluorescence.     

Energetics and product formation by Saccharomyces cerevisiae grown in anaerobic chemostats under nitrogen limitation

Anaerobic fermentation of glucose (20 g/l) by Saccharomyces cerevisiae CBS 8066 was studied in a chemostat (dilution rate = 0.05-0.25 h-1) at different concentrations of the nitrogen source (5.00 g/l or 0.36 g/l ammonium sulphate). The ethanol yield (g ethanol produced/g glucose consumed) was found to be higher and the glycerol yield (g glycerol formed/g glucose consumed) lower during nitrogen limitation than under carbon limitation. The biomass yield on ATP (g dry weight biomass produced/mol ATP consumed), was consequently found to be lower during nitrogen-limited conditions.     

The economic cost of a streptococcal tonsillitis episode

OBJECTIVE: To estimate the cost of a streptococcal tonsillitis episode from the data of a questionnaire. SETTING: Five primary health centres in the west of Sweden. PARTICIPANTS: 101 consecutive patients treated for streptococcal tonsillitis. MAIN OUTCOME MEASURE: The cost estimation included costs for physician visit and drug, travel costs to and from the primary health centre, cost of lost production resulting from the patient's or the guardian's absence from work for physician visit or sick-leave, and cost of telephone consultation with a physician or nurse. RESULTS: The period of illness was on average seven days, time to recovery after treatment five days, and the mean period of sick-leave 2.5 days. The total cost of a tonsillitis episode was about SEK 3,300 (385 USD). Of this sum, the cost for the antibiotic accounted for only 3% and loss of production for 75%. CONCLUSION: Differences in the cost of drugs only have a minor influence on the total cost, while factors causing loss of production, such as efficacy and side effects of the drug, have a greater influence. Economic evaluation of pharmaceuticals will be more relevant in the future, and in the search for the most effective treatment, cost effective studies will be integrated with clinical trials.     

The importance of the Doppler angle

We examined 118 infrainguinal grafts (103 patients) to determine the effect changes in the angle of theta or Doppler angle had on the accuracy of velocity measurements made with a duplex scanner. Four separate measurements of peak velocity were made on each graft. Three measurements were made with the flow toward the probe at 60 degrees, 50 degrees, 120 degrees (60 degrees the other way). The probe was then turned 180 degrees so the flow was away from the probe and measurements were made at 60 degrees. When the primary cursor was aligned with the vessel wall (Technologist A), the mean velocity at 60 degrees was 69.4 cm/sec and 57.3 cm/sec at 50 degrees, a difference of 12 cm/sec. The same measurements done by Technologist B (primary cursor non aligned with the wall), showed a 10 cm/sec difference (60.7 and 50.5). We saw a consistent difference with even this small difference in the angle of theta throughout the study. The mean velocity obtained by Technologist A when the flow was toward the probe was 61.7. When the probe was turned 180 degrees (flow away from the probe), the mean velocity was 60.5. The same measurements by Technologist B were 51.8 and 50.5. This indicates that the direction of the flow of blood towards or away from the probe does not effect the velocity measurements. Using the vessel wall to line up the primary cursor provides a consistent and easily reproducible reference point to compare measurements made on different days but is less important for single measurements.     

Allocation of CO2 emission permits—Economic incentives for emission reductions in developing countries

The economic impacts on developing regions following a global cap and trade system for carbon dioxide are assessed through the use of an energy-economy systems model. Both an equal per capita allocation and a contraction and convergence allocation with convergence of the per capita emissions by 2050 are shown to offer economic incentive for Africa, India and probably also Latin America to accept binding emissions commitments under a 450 ppm carbon dioxide stabilization scenario. The gain for Latin America is mainly a result of increased export revenues from sales of bio-fuels as a result of the climate policy. It is, on the other hand, unlikely that these allocation approaches would offer an economic incentive for China to join the regime because of its high economic growth, present higher per capita emissions than India and Africa, and more costly mitigation options than Latin America. A more stringent allocation for developing countries such as contraction with convergence of the per capita emissions by the end of this century is estimated to generate reduced net gains or increased net losses for the developing regions (though Africa is still expected to gain).