The interaction between applied PEEP and auto-PEEP during one-lung ventilation.

OBJECTIVE: To investigate the relationship between applied external positive end-expiratory pressure (PEEP) and auto-PEEP and the resultant total PEEP experienced by the patient during one-lung ventilation (OLV). DESIGN: A prospective clinical study. SETTING: A university hospital. PARTICIPANTS: Ten adult patients undergoing elective thoracotomies. INTERVENTIONS: End-expiratory airway occlusion and measurement of plateau pressure during two-lung ventilation (TLV) and OLV with and without the application of 5 cm H2O of external PEEP via the anesthetic ventilator. The effect of variation of the inspiratory-expiratory ratio on total PEEP with and without applied external PEEP was also studied. MAIN RESULTS: The mean level (+/-SD) of auto-PEEP changing from two-lung to one-lung ventilation rose from 0.9 (+/-0.8) cm H2O to 6.0 (+/-3.0) cm H2O at an inspiratory-expiratory ratio of 1:2. The application of 5 cm H2O external PEEP did not increase the total PEEP (7.3+/-2.0 cm H2O) significantly. The total PEEP increased significantly when the duration of expiration was decreased, and decreased when expiratory time increased. The change in total PEEP caused by the application of external PEEP during OLV correlated inversely with the preexisting level of auto-PEEP (r=-0.84). CONCLUSION: The change in end-expiratory pressure experienced by the ventilated lung during OLV when external PEEP is applied depends on the preexisting level of auto-PEER This may explain some of the inconsistencies in the clinical results of application of external PEEP during OLV. The total PEEP delivered to the patient should be measured whenever external PEEP is applied during OLV.

Intraoperative complications of laparoscopic cholecystectomy.

We report a series of intraoperative complications of laparoscopic cholecystectomy. Three cases are presented in which subcutaneous emphysema associated with pneumomediastinum, pneumoscrotum, and pneumothorax with pneumomediastinum and ocular emphysema, respectively, developed intraoperatively. These events resulted in no major morbidity to these patients. Use of N2O and monitoring of airway and intraabdominal pressures are discussed.

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.

Enhanced thermodynamic stabilities of yeast iso-1-cytochromes c with amino acid replacements at positions 52 and 102.

We have determined the structures and thermodynamic stabilities of the wild type Asn-52 and unusually thermostable mutant Ile-52 yeast iso-1-cytochromes c (Das, G., Hickey, D. R. McLendon, D., McLendon, G., and Sherman, F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 496-499). Although both structures were similar, Water-166, buried within the wild type protein, is excluded from the Ile-52 mutant, which substantially reorganizes the local hydrogen bonding. Wild type Cys-102 was replaced with alanine or serine to eliminate dimerization in vitro. The Cys-102 (wild type), Ala-102, and Ser-102 proteins were equally stable, whereas the chemically modified Cys-102-SCH3 was less stable. The order of stability observed with replacements at positions 52 and 102 was as follows: Ile-52 Ala-102 greater than Ala-52 Ala-102 greater than Asn-52 Ala-102 ("normal") greater than Gly-52 Ala-102. No significant stabilization was attributed to potential energy interactions expressed as helix-forming propensities of replacements at position 52. A high correlation between differences in free energy changes and transfer free energies suggests hydrophobic interactions are the main factor for enhancing stability in the Ile-52 mutant. Additional possible contributions to the thermostability of the Ile-52 variant are energetic effects due to packing and hydrogen bonding changes surrounding position 52.

Dramatic thermostabilization of yeast iso-1-cytochrome c by an asparagine----isoleucine replacement at position 57.

Two Saccharomyces cerevisiae yeast mutants, cyc1-73 and cyc1-190, contain nonfunctional and presumably unstable forms of iso-1-cytochrome c due to Gly-34----Ser and His-38----Pro replacements, respectively. Second-site reversions that produced Asn-57----Ile replacements at least partially restored function, presumably by alleviating the instability of these two altered iso-1-cytochromes c. Introduction of the Ile-57 replacement by site-directed mutagenesis in an otherwise normal protein resulted in a 17 degrees C increase in the transition temperature (Tm), corresponding to over a 2-fold increase in the free energy change (delta G degrees) for thermal unfolding.

Replacements of lysine 32 in yeast cytochrome c. Effects on the binding and reactivity with physiological partners.

Lysine 32 has been previously implicated by chemical modification and modeling studies as a key component of the domain which controls recognition and binding of cytochrome c to its physiological partners, e.g. cytochrome b2, cytochrome c peroxidase, and cytochrome oxidase. In order to quantitate the importance of this residue, we have investigated the role of Lys-32 in the reactivity of cytochrome c in redox reactions in vitro and in vivo with protein partners by using a series of altered forms of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae in which Lys-32 is replaced by Leu-32, Gln-32, Trp-32, and Tyr-32. Leu-32 and Gln-32 represent substitutions which change charge without seriously affecting the steric bulk of the side chain or the stability of the protein. For the Leu-32- and Gln-32-altered proteins, steady state kinetic studies with cytochrome c peroxidase, cytochrome b2, and cytochrome oxidase showed that neither of the steady state kinetic parameters, Km nor Vmax, were substantially modified by mutation. Studies of single turnover kinetics with a small molecule (ascorbate) or within bound complexes with either cytochrome b5 or cytochrome c peroxidase demonstrated that redox kinetics are only slightly affected by these substitutions. NMR experiments demonstrated that the Gln-32-altered protein can still bind strongly to a physiological partner, cytochrome c peroxidase. Growth in lactate medium demonstrated that the activity in vivo compared with the normal value was reduced to only 85% with the Gln-32- and Leu-32-altered proteins and to 65% with the Trp-32- and Tyr-32-altered proteins. These findings suggest that the evolutionary invariance of Lys-32 reflects only small quantitative changes in the binding and reactivity of cytochrome c.

Thermodynamic stabilities of yeast iso-1-cytochromes c having amino acid substitutions for lysine 32.

Iso-1-cytochromes c having lysine 32 replaced by leucine, glutamine, tyrosine, and tryptophan were prepared from strains of bakers' yeast, Saccharomyces cerevisiae, and chemically blocked at cysteine 107 with methyl methanethiolsulfonate to prevent dimerization. These modified ferricytochromes c were guanidine denatured, and the unfolding thermodynamics were determined by circular dichroism and fluorescence measurements. Thermal unfolding was also monitored by absorbance measurements. The guanidine denaturation midpoints for the altered proteins are smaller than the wild type, while the orders of stability from unfolding free energy changes are: Lys-32 (wild type) approximately Leu-32 approximately Gln-32 (circular dichroism), greater than Gln-32 (fluorescence) greater than Tyr-32 approximately Trp-32. Midpoints and differences in free energy changes for thermal unfolding parallel the fluorescence free energy changes for guanidine-induced unfolding. Thus, the blocked Leu-32 and Lys-32 proteins are equally stable with respect to both chemical and thermal denaturation. The reported data indicate that single replacements may significantly modify protein stability, and that substitution for an evolutionarily retained residue in normal cytochrome c structures does not always destabilize the protein. In addition, in vitro thermal stabilities approximately correlate with in vivo specific activities.

Phenytoin-induced resistance to pancuronium. Use of atracurium infusion in management of a neurosurgical patient.

A case report is presented of a patient receiving chronic phenytoin therapy who demonstrated resistance to pancuronium by increased hourly requirements. Stable neuromuscular blockade was achieved by atracurium infusion at normal rates. Possible explanations for the differences in response to the two non-depolarizing muscle relaxants are discussed.

Comparison of atracurium and succinylcholine for electroconvulsive therapy in a patient with atypical plasma cholinesterase.

Succinylcholine 2-5 mg or atracurium 10-15 mg were given on five separate occasions to a 24-year-old, 64 kg woman homozygous for atypical plasma cholinesterase who was undergoing electroconvulsive therapy (ECT). Atracurium blockade was reversed with atropine, 0.6 mg and edrophonium, 35 mg. Train-of-four stimulation was applied to the ulnar nerve and the force of contraction of the adductor pollicis muscle was recorded. Doses producing 90 per cent first twitch blockade were 2.5 and 15 mg for succinylcholine and atracurium respectively. The onset of action was 6 min for both relaxants, and time to 90 per cent first twitch recovery was 20 min for succinylcholine and 16 min for the atracurium-edrophonium combination. It is concluded that the use of atracurium in these patients does not offer marked advantages over small doses of succinylcholine.

Replacement of the invariant lysine 77 by arginine in yeast iso-1-cytochrome c results in enhanced and normal activities in vitro and in vivo.

Oligonucleotide-directed mutagenesis of the yeast Saccharomyces cerevisiae was used to generate an abnormal iso-1-cytochrome c having an Arg-77 replacement of the normal Lys-77; this Lys-77 residue is evolutionarily conserved in most eukaryotic cytochromes c and is trimethylated in fungal and plant cytochromes c. Examination of strains having a single chromosomal copy of the gene encoding the Arg-77 protein indicated that the altered protein was synthesized at the normal rate and that it had normal or near normal activity in vivo. Examination of enzymatic activities in vitro with cytochrome b2, cytochrome c peroxidase, and cytochrome c oxidase indicated that the altered iso-1-cytochrome c has equal or enhanced catalytic efficiencies. Thus, replacement of the evolutionarily conserved residue Lys-77 produces no or only minor effects both in vivo and in vitro.

Hyperkalaemic cardiac arrest following intravenous hydralazine and propranolol therapy post-embolectomy.

A case of hyperkalaemic cardiac arrest occurring in a 50-year-old woman after bilateral femoral embolectomies is presented. The postoperative course was complicated by acute renal failure, metabolic acidosis, hypotension and low cardiac output. The possible contribution of hydralazine and propranolol therapy to the rapid development of hyperkalaemia is discussed.

Effects of terminal mismatches on RNA stability: thermodynamics of duplex formation for ACCGGGp, ACCGGAp, and ACCGGCp.

The thermodynamics of ACCGGCp, ACCGGAp, and ACCGGGp helix formation have been measured in 1 M NaCl. The terminal mispairs stabilize the CCGG core duplex at 10(-4) M in the order AC approximately equal to AA less than AG approximately equal to AU. The data reported provide thermodynamic parameters for RNA structure prediction and suggest useful approximations for including other mismatches in current algorithms.

Solvent effects on the stability of A7U7p.

The thermodynamics of double-helix formation were measured spectrophotometrically for A7U7 in water at 1 M NaCl and for A7U7p in a variety of solvent mixtures and salt. Comparison of the A7U7 results with calorimetric measurements indicates duplex formation involves intermediate states. For A7U7p between 0.06 and 0.55 M Na+, dTm/d(log [Na+]) = 17.4 degrees C, similar to the value of 19.6 degrees C for poly-(A).poly(U) [Krakauer, H., & Sturtevant, J. M. (1968) Biopolymers 6, 491-512]. At 1 M NaCl, the A7U7p duplex is most stable in 100% water. For 10 mol % solutions, the order for A7U7p duplex stability is ethylene glycol greater than glycerol greater than ethanol greater than 2-propanol greater than dimethyl sulfoxide greater than 1-propanol greater than formamide greater than N,N-dimethylformamide greater than urea greater than dioxane. Comparison of changes in stability and thermodynamic parameters with literature results for proteins suggests proteins and A7U7p interact differently with solvent. The results suggest hydrophobic bonding is not a major contributor to the stability of the A7U7p duplex. Comparisons with bulk solvent surface tension suggest the energy of cavity formation is also not a major contributor to duplex stability.

Thermodynamic studies of RNA stability.

Enthalpies and entropies of helix stabilization due to addition of 3' terminal unpaired nucleotides to a CCGG or GGCC core double helix are derived from UV melting studies. The results suggest stacking provides a significant fraction of the free energy of a terminal base pair. The effects of temperature, aggregation, and ionic strength on the determination of thermodynamic parameters are considered. Helix propagation parameters are revised and extended based on recent additions to the data set.