Prayer in medicine: a survey of primary care physicians.

Prayer and spirituality have been shown to have a significant impact on several health variables. Additionally, studies have shown that patients think prayer is important to their health. Very little research, however, has been done to determine primary care physicians' opinions regarding prayer and spirituality as it pertains to healthcare. We surveyed primary care physicians in Mississippi to assess their use of prayer in medical practice. Ninety-one percent of respondents considered prayer an important treatment modality, but 50.6% rarely or never discussed prayer with patients. Most who excluded prayer from clinical practice did so to avoid imposing their beliefs upon patients. A majority of primary care physicians in Mississippi recognize prayer as an important psychosocial variable in assessing and treating patients, but many are hesitant to incorporate this variable into the doctor-patient encounter.

Cleavage of tubulin by vanadate ion.

Vanadate is known to cleave proteins in a near-uv-dependent manner. We have found that vanadate will cleave alpha- and beta-tubulin upon photoirradiation (419 nm emission maxima) under conditions when tetravanadate, pentavanadate, and decavanadate are in solution. The reaction is independent of GTPMg or GDPMg, and cleavage occurs at two or more sites per chain. Cleavage was studied at pH 6.0 (2(N-morpholino)ethanesulfonic acid (Mes) and phosphate), pH 6.9 (piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes)), pH 7.0 (phosphate), and pH 8.0 (N-(2-hydroxyethyl)piperazine-N'-bis(2-ethanesulfonic acid) (Hepes) and phosphate). The concentration of vanadate oligomer species, as determined by 51V NMR, was correlated with the extent of cutting. In organic buffers, low pH and high vanadate concentration favored oligomer formation, especially tetra and decavanadate. In phosphate buffer at pH 7 and 8, decamer is more prevalent, and at pH 6, phosphate buffer appears to favor a different oligomer form, V', appearing at -582 ppm. Cleavage is best correlated with the presence of cyclic tetravanadate at pH 6.9 in Pipes buffer and the V' species at pH 6.0 in phosphate buffer. Cleavage efficiency is also affected by interactions of photoactivated vanadate species with organic buffer components. In phosphate buffer no photochemical degradation of vanadate species occurs. Analysis using sodium dodecyl sulfate (SDS) gel electrophoresis and western blotting showed that vanadate produced cleavage patterns and nonenzymatic cleavage patterns resulting from boiling tubulin in SDS sample buffer (J. J. Correia, L. D. Lipscomb, and S. Lobert, 1993, Arch. Biochem. Biophys. 300, 105-114) are not the same. Attempts to identify the locations of the vanadate cleavage sites on the protein through N-terminal sequencing was unsuccessful, apparently due to the presence of blocked amino groups. We conclude that tetravandate cleaves tubulin upon photoirradiation, that organic buffers can interact with vanadate oligomers upon photoirradiation, and that in phosphate buffer photocleavage is enhanced by an absence of photochemical degradation and a preference for forming photoactive vanadate oligomers. These results have general application to photoirradiation studies of any protein in the presence of vanadate.

Nondisulfide crosslinking and chemical cleavage of tubulin subunits: pH and temperature dependence.

Tubulin is known to be extremely unstable. The denaturation process partially involves irreversible aggregation, mediated by disulfide crosslinking. In addition, tubulin is known to undergo chemical cleavage during boiling in sodium dodecyl sulfate (SDS), a process that generates small peptides that have been mistaken for low molecular weight MAPs. Similar peptide cleavage has now been observed during two-dimensional denaturing isoelectric focusing-SDS-polyacrylamide gel electrophoresis. This phenomenon has complicated interpretation of limited proteolysis studies of tubulin by subtilisin. In an effort to avoid this problem we have undertaken a detailed study of the solution conditions that promote chemical cleavage of tubulin. The cleavage reaction is found to be strongly pH, time, and temperature dependent. Nondenatured and denatured tubulin is susceptible to peptide cleavage, suggesting that primary structure is more important than secondary structure in selection of susceptible bonds. After transfer of cleavage products from an SDS gel to a polyvinylidene difluoride membrane, amino acid sequencing has confirmed cleavage at Asp-Pro bonds, at position 306 in alpha-tubulin and at position 304 in beta-tubulin. We also infer cleavage at the only additional Asp-Pro peptide bond located at position 31 in beta-tubulin. Heat-induced cleavage at Asp-Pro accounts for 5 of the 13 bands observed on SDS gels. In addition, a minor alpha-tubulin band has been sequenced from a two-dimensional gel, corresponds to cleavage at Asp-Cys located at alpha-tubulin position 200, and accounts for two additional bands observed on SDS gels. Under nondenaturing and nonpolymerizing conditions tubulin undergoes extensive intermolecular, disulfide crosslinking. At elevated temperatures and high pH, a small fraction of the crosslinking is not reduced by beta-mercaptoethanol. Disulfide-crosslinked aggregates are not suspected because carboxymethylation of tubulin does not prevent their formation. Lysinoalanine has been found by amino acid analysis and thus covalent lysine-dehydroalanine crosslinks are suspected. Dehydroalanine is formed by beta-elimination at serine and thus the presence of lysinoalanine is consistent with cleavage at Gly-Ser peptides, the most unstable serine peptide bond, and accounts for most of the remaining cleavage data.