Australian and New Zealand Anaesthetic Allergy Group Perioperative Anaphylaxis Investigation Guidelines.

These guidelines are a consensus document developed by a working party of the Australian and New Zealand Anaesthetic Allergy Group (ANZAAG) to provide an approach to the investigation of perioperative anaphylaxis. They focus primarily on the use of skin testing as it is the investigation with the greatest clinical utility for the identification of the likely causative agent and potentially safer alternatives. The practicalities and process of skin testing, its limitations, and the place of other tests are discussed. These guidelines also address the roles of graded challenge and in vitro testing. The implications of anaphylaxis associated with neuromuscular blocking agents, beta-lactam antibiotics, local anaesthetic agents and chlorhexidine are discussed. Evidence for the recommendations is derived from literature searches using the words skin test, allergy, anaphylaxis, anaesthesia, and each of the individual agents listed in these guidelines. The individual articles were then reviewed for suitability for inclusion in these guidelines. Where evidence was not strong, as is the situation for many perioperative agents, expert consensus from the ANZAAG working party was used. These guidelines are intended for use by specialists involved in the investigation of perioperative allergy. They have been approved following peer review by members of ANZAAG and are available on the ANZAAG website: http://www.anzaag.com/anaphylaxis-management/testing-guidelines.pdf.

Age-related changes in bovine alpha-crystallin and high-molecular-weight protein.

The high-molecular-weight (HMW) protein from the lens is composed mostly of alpha-crystallin in a highly aggregated state. Bovine HMW protein was carefully separated from alpha-crystallin by size-exclusion chromatography. alpha-Crystallin has chaperone-like ability whereby it stabilizes other proteins under conditions of stress (e.g. heat). Comparison of bovine HMW protein and alpha-crystallin shows that the HMW protein has a markedly reduced chaperone ability compared to alpha-crystallin. However, in contrast to the results of other workers, we observe no alteration with age in the ability of alpha-crystallin to act as a chaperone. Using electrospray ionisation mass spectrometry, changes in the phosphorylation of the alpha-crystallin subunits with age have been quantified. Phosphorylation of alpha-crystallin occurs early in life but does not alter in proportion after about three years of age. In addition, phosphorylation of the A subunit of alpha-crystallin has little effect on its chaperone ability. As is found in the artificially prepared HMW complex of alpha- and gamma-crystallin, NMR spectroscopy shows that in the naturally occurring HMW protein, the short C-terminal extension of the alpha B subunit has lost its flexibility whereas the alpha A subunit extension is still flexible. Post-translational modifications therefore seem to have little effect on the chaperone action of alpha-crystallin, but alterations in the quaternary structure of alpha-crystallin via incorporation into the HMW aggregate, lead to major changes in the chaperone ability of the protein. The results are consistent with the notion that one of the contributing factors to cataract formation in the lens is the depletion of alpha-crystallin with age as it is converted into the HMW protein.

On the interaction of alpha-crystallin with unfolded proteins.

alpha-Crystallin, a major protein component of the lens, has chaperone-like properties whereby it prevents destabilised proteins from precipitating out of solution. It does so by forming a soluble high-molecular-weight (HMW) complex. A spectroscopic investigation of the HMW complex formed between a variety of unfolded proteins and bovine alpha-crystallin is presented in this paper. As monitored by fluorescence spectroscopy, a large amount of the hydrophobic probe, 8-anilino-1-naphthalene sulfonate (ANS) binds to the HMW complex implying that the complexed proteins (alcohol dehydrogenase (ADH), gamma-crystallin and rhodanese) are bound in an unfolded, possibly molten-globule state. The interaction between the anionic surfactant, sodium dodecyl sulfate (SDS) and ADH at high temperatures gives rise to a similar large increase in ANS fluorescence to that for the complex between alpha-crystallin and ADH. SDS, like alpha-crystallin, therefore complexes to proteins in their unfolded state leaving a large hydrophobic surface exposed to solvent. Unlike other chaperones (e.g., GroEL, DnaK and SecB), alpha-crystallin does not interact with unfolded, hydrophobic but stable proteins (e.g., reduced and carboxymethylated alpha-lactalbumin and alpha-casein). It is concluded that alpha-crystallin will only complex with proteins that are about to precipitate out of solution, i.e., ones that are severely compromised. 1H-NMR spectroscopy of the HMW complex formed between alpha-crystallin and gamma-crystallin indicates that the short C-terminal extension of alpha B-crystallin, but not that of alpha A-crystallin, has lost its flexibility in the complex implying that the former is involved in interactions with the unfolded gamma-crystallin molecule, possibly electrostatically via its two C-terminal lysine residues.