X-ray structural analysis of the yeast cell cycle regulator Swi6 reveals variations of the ankyrin fold and has implications for Swi6 function.

Swi6 is a 92,000 Mr protein common to two distinct transcriptional activation complexes (SBF and MBF) that coordinate gene expression at the G1-S boundary of the yeast cell cycle. The X-ray structure of a central 36,000 Mr fragment has been determined and refined at 2.1 A resolution. The structure reveals a basic framework of five ankyrin repeat modules that is elaborated through a series of helical insertions distinguishing it from structures of other ankyrin repeat proteins. A second domain contains an approximately 30-residue region of extended structure that interacts with the ankyrin repeat core over a substantial proportion of its surface. Conservation of residues buried by these interactions indicates that all members of the Swi6/Cdc10 family share a similar architecture. Several temperature-sensitive mutations within Swi6 and Cdc10 appear to disrupt these interdomain contacts rather than destabilize the ankyrin repeat core. The unusual domain arrangement may be crucial for the modulation of interactions with other co-regulatory molecules such as cyclin-CDK complexes, and has implications for the quaternary interactions within the multisubunit SBF and MBF transcription complexes.

Structural and functional architecture of the yeast cell-cycle transcription factor swi6.

The structural and functional organisation of Swi6, a transcriptional regulator of the budding yeast cell cycle has been analysed by a combination of biochemical, biophysical and genetic methods. Limited proteolysis indicates the presence of a approximately 15 kDa N-terminal domain which is dispensable for Swi6 activity in vivo and which is separated from the rest of the molecule by an extended linker of at least 43 residues. Within the central region, a 141 residue segment that is capable of transcriptional activation encompasses a structural domain of approximately 85 residues. In turn, this is tightly associated with an adjacent 28 kDa domain containing at least four ankyrin-repeat (ANK) motifs. A second protease sensitive region connects the ANK domain to the remaining 30 kDa C-terminal portion of Swi6 which contains a second transcriptional activator and sequences required for heteromerisation with Swi4 or Mbp1. Transactivation by the activating regions of Swi6 is antagonised when either are combined with the central ankyrin repeat motifs. Hydrodynamic measurements indicate that an N-terminal 62 kDa fragment comprising the first three domains is monomeric in solution and exhibits an unusually high frictional coefficient consistent with the extended, multi-domain structure suggested by proteolytic analysis.

Effect of osmolytes on the exchange rates of backbone amide protons in proteins.

Osmolytes are small organic solutes produced by the cells of all organisms (except halobacteria) in high stress situations (e.g. extremes of salt concentration, high temperature, etc.) to stabilize their macromolecules and so conserve biological activity. They do not interact with the macromolecule directly but act by altering the solvent properties in the cellular environment, and so their presence indirectly modifies the stability of proteins. In this paper we examine the effect of a model osmolyte, glycine, on the stabilization of two proteins, chymotrypsin inhibitor 2 and horse heart cytochrome c. We have used NMR to monitor the effect of this osmolyte on amide hydrogen exchange rates, which allows a probe at discrete points within the protein structure. Hydrogen exchange rates of specific backbone amide protons provide information about the localized structural fluctuations that expose these amides to solvent and allow exchange to take place. We find that the presence of a high concentration of glycine osmolyte has a profound stabilizing effect on the proteins studied, which is accompanied by a large reduction of the exchange rate constants of most slowly exchanging amide protons. The spectra indicate that this arises without significant changes in the three-dimensional structure. However, the effects on individual amide protons within a single protein were not uniform, and a wide variation in the magnitude of the effects was observed. This ranged from no apparent change in the exchange rate, to decreases in the exchange rate constant by over 2 orders of magnitude. The osmolyte appears to alter a number of different processes that contribute to the observed exchange rates, and no simple generalization allows prediction of the extent of stabilization at any individual location. The results are discussed in light of the available structures of the proteins studied.

Inhaled lysine-aspirin as a bronchoprovocation procedure in aspirin-sensitive asthma: its repeatability, absence of a late-phase reaction, and the role of histamine.

Inhalation of an aerosolized solution of lysine-aspirin has previously been described as a safer technique than oral challenge with aspirin for the diagnosis of aspirin-sensitive asthma. We describe a modification of this method that involves inhalation of serially doubling incremental concentrations of lysine-aspirin by a standardized technique and allows construction of concentration-response curves. In 11 subjects with asthma, mean (SEM) age 48.2 (2.9) years, the geometric mean (range) provocation concentrations of histamine and lysine-aspirin required to produce a 20% decrease in FEV1 from baseline were 0.6 (0.04 to 3.2) and 48.3 (15.5 to 219) mg/ml, respectively. No relationship was found between these values. In seven of nine subjects investigated on two consecutive occasions, bronchoconstriction with lysine-aspirin was repeatable to within a single doubling concentration difference. Bronchoconstriction provoked by lysine-aspirin was more rapid than with oral aspirin and was not followed by any late asthmatic reaction or increase in nonspecific airway hyperresponsiveness. In six subjects, premedication with the selective H1 histamine-receptor antagonist, terfenadine, had no significant effect on bronchoconstriction provoked by inhaled lysine-aspirin, indicating little role for release of histamine in the response. We conclude that inhalation of lysine-aspirin may be used as a bronchoprovocation procedure for the diagnosis and investigation of aspirin-sensitive asthma.

Aspects of clinical trials with ceftazidime worldwide.

Overall results from worldwide clinical studies of ceftazidime in open and comparative trials conducted over a four-year period in North America, Europe, and elsewhere are presented. Data from 3,570 patients treated with ceftazidime in open studies and from 1,340 patients receiving ceftazidime and 1,180 patients receiving other antibiotics in comparative studies are discussed. The comparative antibiotics consisted of aminoglycosides alone in 14.2 percent of patients, aminoglycosides combined with one or two beta-lactams in 43 percent, beta-lactams alone in 34 percent, and other antibiotic combinations in 8.8 percent. In comparative studies, bacterial clearance rates were 80.6 percent for ceftazidime and 72.5 percent for other antibiotics. Clinically, 92.6 percent of the infected sites in ceftazidime-treated patients in open studies were cured or improved, and 91.1 percent were cured or improved in comparative studies; 84.8 percent of the infected sites were cured or improved in patients treated with other antibiotics. Data indicate that ceftazidime monotherapy is as effective as combination antibiotic therapy in the empiric treatment of febrile non-neutropenic patients. The role of ceftazidime monotherapy in the treatment of febrile neutropenic patients is not yet firmly established. Superinfections were reported in 2.6 percent of the patients treated with ceftazidime alone in open trials, in 4.2 percent of the patients treated with ceftazidime alone in comparative trials, and in 8.5 percent of the patients treated with other antibiotics. During treatment, the sensitivity of bacteria to ceftazidime decreased in 2.5 percent of the pathogens isolated. Adverse events occurred in 10.2 percent of patients treated with ceftazidime in open studies, in 8.4 percent of those treated with ceftazidime in comparative studies, and in 8.8 percent of those treated with other antibiotics. The adverse events in both ceftazidime- and control-treated patients were thought by the investigators to be drug-related in about 40 percent of cases, not drug-related in 20 percent of cases, and of unknown etiology in the remaining cases.

Precise comparison of experimental and theoretical SNRs in CO2 laser heterodyne systems.

A detailed comparison of experimental and theoretical SNR in an IR laser heterodyne system has been made with three different signal analyzers. Good agreement, considerably better than a factor of 1.5, is reported. Accurate allowance was made for transmission in the receiver optics, the effective quantum efficiency of the detector due to shot noise domination by the local oscillator, and for coherent speckle effects across the collection aperture. The evaluation of SNR with a surface acoustic wave spectrum analyzer and digital integrator is described in some detail. As an illustration an absolute measurement of backscattering strength in the atmosphere from an airborne equipment at altitudes up to 13.1 km is provided.

Ceftazidime: aspects of efficacy and tolerance.

Clinical trial results have been analysed from 2607 patients (2953 infections, of which 38% were severe) treated with ceftazidime alone and from 466 patients (507 infections - 24% severe) treated with one or more comparative antibiotics. Of 235 staphylococcal infections (all sites) treated with ceftazidime alone 84% cleared and 16% relapsed or failed; of 28 infections treated with other antibiotics, 82% cleared and 18% relapsed or failed. Of 541 pseudomonas infections (all sites) treated with ceftazidime 60% cleared and 40% relapsed or failed; of 76 infections treated with other agents 43% cleared and 57% relapsed or failed. Adverse events were seen in 8.9% of the 2,607 ceftazidime treated and in 8.2% of the 466 patients treated with other agents and included local and gastrointestinal intolerance and hypersensitivity reactions. Changes in laboratory tests included eosinophilia, Coombs' positivity and liver enzyme increases. Superinfection occurred in 2.5% of patients treated with ceftazidime and in 5.2% of patients treated with other antibiotics. Deaths from all causes during or shortly after treatment were reported in 3.3% of the 2607 ceftazidime-treated and in 5.8% of the 466 patients treated with other agents. Factors, including neutropenia, influencing these differences are analysed.

Effect of timing of cefuroxime dosage on its protection of rats against gentamicin nephrotoxicity.

The neophrotoxic effect on rats of once daily treatment with 40 or 45 mg gentamicin/kg/day for ten days was substantially reduced by administration of 4 g cefuroxime/kg/day, either at the same time or eight hours later. This dosage of cefuroxime was protective when given for only two consecutive days starting on the first to third days of gentamicin treatment, but enhanced gentamicin nephrotoxocity when started on the sixth or subsequent days.

Cefuroxime: human pharmacokinetics..

Single doses of cefuroxime, a new parenteral cephalosporin, were given to 44 normal male subjects. Doses of 0.25, 0.5, 0.75, or 1.0 g were given intramuscularly to 33 volunteers. Mean peak serum concentrations of 14.8, 25.7, 34.6, and 40.0 mug/ml were assayed at 29 to 45 min, and measurable levels were present 8 h after the 0.5-g and higher doses. Single intravenous bolus doses of 0.25, 0.5, or 1.0 g were given to nine volunteers, and mean levels of 39, 66, and 99 mug/ml were assayed at 3 min. The antibiotic has a mean ultimate serum half-life of 70 min, a mean serum protein binding of 33%, a metabolic stability of greater than 95%, an apparent distribution volume of 11.1 to 15.8 liters/1.73 m(2) depending on dose, and a mean urinary recovery of at least 95% for all parenteral doses. The cefuroxime/creatinine clearance ratios for all volunteers indicated that 43 to 54% of the antibiotic is secreted through the kidney tubules, and this was confirmed in some volunteers who received probenecid simultaneously. The injections by both routes were well tolerated, and the slight pain experienced after intramuscular injection disappeared within a few minutes. Physical and laboratory investigations in the volunteers showed that administration of cefuroxime had had no adverse effects. There was no evidence of absorption after oral administration.

Cefuroxime - a new cephalosporin antibiotic.

Cefuroxime is a new broad spectrum cephalosporin antibiotic for administration by injection. It is stable to most beta-lactamases. It is active against gram-positive organisms, including penicillinase-producing staphylococci, and has wide activity against gram-negative bacilli including Enterobacter and many strains of indole-positive Proteus spp. The substance is also highly active against Haemophilus influenzae and Neisseria gonorrhoeae. Studies on human volunteers showed that it produced high, long-lasting blood levels with virtually complete recovery of unchanged antibiotic in the urine. No evidence of toxicity due to cefuroxime was found. Slight, short-lived pain followed intramuscular injection, and the compound was well tolerated intravenously.