Generalised bilayer perturbation from peptide helix dimerisation at membrane surfaces: vesicle lysis induced by disulphide-dimerised melittin analogues.

The effects of covalent dimerisation of melittin by disulphide formation in cysteine-substitution analogues, (melittin K23C)2 and (melittin K23Q,Q25C)2, on the kinetics of pore formation in phosphatidylcholine small unilamellar vesicles was measured under low ionic strength conditions. The initial rate of melittin-induced pore formation increased with the square of the peptide concentration, whereas both disulphide-dimerised melittin analogues showed a first-order dependence of pore formation rates on peptide concentration. These results indicate that peptide dimerisation is rate-limiting for pore formation under these conditions. A model for a generalised bilayer perturbation resulting from the self-association of a pair of peptide helices at the membrane surface is proposed which may have implications for a number of biological processes that involve the interaction of helical polypeptides with membranes.

From system changes to workplace accommodation: rehabilitation trends in training, education, and the system of professions in Australia.

The first part of this paper highlights the developments in rehabilitation education and training which have occurred as a result of system changes to workers' compensation and rehabilitation in Australia. Each system change has ushered in new responses to the question of what knowledge, attitudes and skills are required by rehabilitation service delivery personnel, responses which appear to trace an identifiable direction towards a greater concern for professional education. A more silent development, occurring in parallel to workers' compensation and rehabilitation changes and to directions in education and training, has been the emergence of rehabilitation counselling as a relatively new player in the rehabilitation field. The second part of this paper presents preliminary findings of a study which indicate a change in the division of labour in the rehabilitation field in Australia, reinforcing the importance of rehabilitation counselling activities.

Endoscopic technique to mark the site of tracheal stenosis for resection.

BACKGROUND: It is difficult to precisely localise the extent of the diseased segment on the external aspect of a stenotic trachea. A technique has been developed of marking the upper margin of stenosis, in order to open the airway at the appropriate level during segmental resection. MATERIALS AND METHODS: Prior to the open reconstructive procedure, the stenosis is visualised using microlaryngoscopy. An endo-extraluminal technique is used to drive a suture from inside out through the skin; this then serves to mark the exact top margin of the stenotic segment. This suture serves as a guide for the surgeon during the open approach to tracheal resection. RESULTS: This technique was performed in 16 cases, and allowed precise localisation of the stenosis in each case. CONCLUSION: Transcutaneous localisation of laryngotracheal stenosis, using the Lichtenberger device, is an easy and reliable technique requiring a minimum of additional time.

Voice quality improvement after management of unilateral vocal cord paralysis with different techniques.

The aim of this study was to objectively evaluate the voices of patients suffering from unilateral vocal cord paralysis, before and after endoscopic augmentation and thyroplasty. In the past, we used injectable Teflon to treat this condition; later techniques included collagen injection and Isshiki thyroplasty. In the last 7 years, preferred treatment methods have included Bioplastique injection and lipoaugmentation of the vocal cords as well as medialization thyroplasty using a titanium implant according to Friedrich. Pre- and postoperative data was evaluated and compared to 25 patients. Appropriate glottic closure of the vocal cords was achieved in every case, in most cases after the first intervention. We used voice range profile measurements to evaluate the results. An objective evaluation was performed using the Friedrich dysphonia index. Significant improvements were found: the dysphonia index decreased in every case, from an average of 2.47, preoperatively, to an average of 1.18 postoperatively. In agreement with earlier studies, voice pitch range was the only parameter that not significantly improved. There was no statistical difference between the lipoaugmentation and thyroplasty according to Friedrich. We concluded that both endoscopic methods and thyroplasty can be used to achieve an optimal result. Cases must be evaluated individually so that the best technique, or combination of methods can be determined.

Crystallization of redox-insensitive Oct1 POU domain with different DNA-response elements.

The POU domain of the human Oct1 transcription factor has been crystallized with two different POU-dimer-binding DNA elements. Protein-DNA cocrystals suitable for structural analysis could be obtained only with a redox-insensitive version of the POU domain. The recombinant protein expression in a prokaryotic host was adjusted for fast purification. Optimized crystals were obtained by systematically varying the length of the oligonucleotide and by modifying cryofreezing procedures. These steps are generally applicable to the preparation of protein-DNA complexes for structural studies.

Self-association of disulfide-dimerized melittin analogues.

Two cysteine substitutions of bee venom melittin have been synthesized to investigate the effects of disulfide cross-linking on the self-association properties of the peptide in solution. K23C melittin (mltK23C) was designed to link nonpolar surfaces of the amphipathic melittin helix on the basis of the close juxtaposition of pairs of K23 side chains in the crystal of the native melittin tetramer. K23Q/Q25C melittin (mltQ25C) was designed to link the polar surfaces of the peptide such that self-association in membrane bound states might be stabilized. The mltK23C disulfide dimer, (mltK23C)2, is highly structured at low pH under conditions where native melittin, and the mltK23C monomer, are unstructured. High-resolution NMR, circular dichroism, and fluorescence spectroscopy established that (mltK23C)2 is a helical monomer (pseudodimer) with stable helical segments between residues 2-13 and 15-25. Although the symmetrical nature of the pseudodimer prevented high-resolution structure determination, analysis of calculated hydrogen bond lengths, chemical shifts, near-UV circular dichroism, and urea denaturation demonstrated similarities with alpha-helical coiled coils and with the structure of native melittin in methanol. Stopped flow fluorescence showed that (mltK23C)2 underwent pH- and divalent anion-linked dimerization to a melittin-like pseudotetramer, indicating that a pair of disulfide bonds could be accommodated in a structure similar to the native melittin crystal structure. Despite incorporation of two disulfide bonds into the melittin tetramer, the folding free energy (DeltaGw) of [(mltK23C)2]2 was similar to that for the native melittin tetramer under the condition used. Incorporation of a disulfide bond on the polar helix face in melittin did not stabilize helical structure in the absence of self-association. Instead, this molecule underwent pH- and divalent anion-linked self-association to an ill-defined aggregate which precipitated.

Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated by a specific POU dimer configuration.

POU domain proteins contain a bipartite DNA binding domain divided by a flexible linker that enables them to adopt various monomer configurations on DNA. The versatility of POU protein operation is additionally conferred at the dimerization level. The POU dimer formed on the PORE (ATTTGAAATGCAAAT) can recruit the transcriptional coactivator OBF-1, whereas POU dimers formed on the consensus MORE (ATGCATATGCAT) or on MOREs from immunoglobulin heavy chain promoters (AT[G/A][C/A]ATATGCAA) fail to interact. An interaction with OBF-1 is precluded since the same Oct-1 residues that form the MORE dimerization interface are also used for OBF-1/Oct-1 interactions on the PORE. Our findings provide a paradigm of how specific POU dimer assemblies can differentially recruit a coregulatory activity with distinct transcriptional readouts.

Differential dimer activities of the transcription factor Oct-1 by DNA-induced interface swapping.

Two crystal structures of Oct-1 POU domain bound to DNA provide a rationale for differential, conformation-dependent recruitment of transcription cofactors. The POU-homeo and POU-specific subdomains of Oct-1 contain two different nonoverlapping pairs of surface patches that are capable of forming unrelated protein-protein interfaces. Members of the POU factor family contain one or two conserved sequence motifs in the interface that are known to be phosphorylated, as noted for Oct-1 and Pit-1. Modeling of Oct-4 reveals the unique case where the same conserved sequence is located in both interfaces. Our studies provide the basis for two distinct dimeric POU factor arrangements that are dictated by the architecture of each DNA response element. We suggest interface swapping in dimers could be a general mechanism of modulating the activity of transcription factors.