One hospital's experience with a "Do not resuscitate" policy.

A "No not resuscitate" policy was instituted at McMaster University Medical Centre, Hamilton, in January 1979. Its objectives were to ensure that physicians decide on the appropriateness of resuscitation attempts before they might be needed; to have each physician consult his or her patients, or the families of incompetent patients, to determine their wishes concerning further treatment; and to provide legal protection of or physicians and the hospital in regard to the policy. To determine the effectiveness of the "Do not resuscitate" policy a questionnaire was sent to a sample of the professional staff of the hospital; the overall response rate was 87%. The respondents felt that a better way of informing hospital staff of the policy and its objectives was needed. However, the results of the questionnaire suggested that, on the whole, the policy was perceived as beneficial to both patients and physicians at the hospital.     

Is the “Difference index” useful for assessment of protein relatedness?

This paper assesses the usefulness of the Difference Index (Metzger, Shapiro, Mosimann &; Vinton, 1968) for predicting, from amino acid compositions, whether two proteins are related or unrelated. It is concluded that, with a 5 % probability of false identification, Difference Index values less than 10-0 indicate relatedness and values greater than 26·8 indicate unrelatedness. A large proportion of protein pairs have Difference Index values in the region 10·0–26·8 and cannot therefore be reliably identified as related or unrelated by this criterion.     

Water-soluble (1→3), (1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure

Water-soluble (1→3),(1→4)-β-d-glucans isolated from barleys grown in Australia and the UK were depolymerised using a purified (1→3),(1→4)-β-d-glucan 4-glucanohydrolase (EC 3.2.1.73). Oligomeric products were quantitatively separated by high resolution gel filtration chromatography and their structures defined by methylation analysis. Approximately 90% (w/w) of each polysaccharide consists of cellotriosyl and cellotetraosyl residues separated by single (1→3)-linkages but blocks of 5–11 (1→4)-linked glucosyl residues are also present in significant proportions. Periodate oxidation followed by Smith degradation suggested that contiguous (1→3)-linked β-glucosyl residues are either absent, or present in very low frequency. The potential for misinterpretation of data due to incomplete Smith degradation was noted.The irregularly-spaced (1→3)-linkages interrupt the relatively rigid, ribbon-like (1→4)-β-glucan conformation and confer a flexibility and ‘irregular’ shape on the barley (1→3),(1→4)-β-d-glucan, consistent with its solubility in water. Molecular models incorporating the major structural features confirm that the polysaccharide is likely to assume a worm-like conformation in solution. Non-covalent interactions between long blocks of (1→4)-linkages in (1→3),(1→4)-β-d-glucans, or between these blocks and other polysaccharides, offer a possible explanation for the organisation of polysaccharides in the framework of the cell wall.     

Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa

The highly conserved, buried, Asp 26 in Escherichia coli thioredoxin has a pKa = 7.5, and its titration is associated with a sizable destabilization of the protein [Langsetmo, K., Fuchs, J., & Woodward, C. (1991) Biochemistry (preceding paper in this issue)]. A fit of the experimental pH dependence of thioredoxin stability to a theoretical expression for the pH/stability relation in proteins agrees closely with a pKa value of 7.5 for Asp 26. The agreement between the experimental and theoretical changes in protein stability due to substitution of Asp 26 by alanine is also good. The local structure in the vicinity of Asp 26 in the low-pH crystal structure (with uncharged Asp 26) is hydrophobic, indicating that the aspartate would be highly destabilized. In theoretical calculations, the desolvation penalty for deprotonating Asp 26 in this environment is similar to the total protein folding energy. As a consequence, the Asp 26 pKa would be much greater than 7.5, and/or the protein might not fold. This suggests that a compensating process partially stabilizes the Asp 26 carboxyl group when it is charged. A simple model for this proposed, whereby the Lys 57 side chain rotates to form a salt bridge with Asp 26 when it is deprotonated.     

In vivo recovery of muscle contraction after alpha-bungarotoxin binding

Acetylcholine receptors were inactivated in vivo at the mouse neuromuscular junction using alpha-bungarotoxin (alpha-BTX). It was found that neurally produced muscle contraction recovered within 4-8 days (halftime similar to 3 days). Actinomycin D interfered with this recovery, but did not affect normal nerve-stimulated muscle contraction. If the response was initially eliminated by [125-I]alpha-BTX and the end plates examined by EM autoradiography, no evidence of mass internalization of bound radioactivity during recovery was seen. The fine structure of the end plates and muscle was unaltered during the post-alpha-BTX recovery period.     

Electrical Signs of New Membrane Production during Cleavage of Rana pipiens Eggs

Rana pipiens eggs dividing normally in diluted Ringer's solution show an increase in transmembrane potential inside negative, a decrease in resistance, and no change in total surface membrane capacitance at the appearance of a division furrow. Furrows of eggs in solutions with the tonicity of full Ringer develop partially, then regress so that the surface is again spherical. The potential and resistance changes are greater and substantial increases in capacitance occur when furrowing is so inhibited. It is proposed that the electrical changes at division are due to the introduction of new plasma membrane, between the blastomeres, having selective permeability to K and a low resistance compared to the outer spherical membrane. A narrow gap between blastomeres limits current flow through new membrane during normal division. A direct exposure of new membrane to the bathing medium when furrowing is disrupted results in larger changes in potential and resistance and permits the capacitance of new membrane to be detected.     

Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.