Aspects of the hemes and modulation of hydrogen donors in catalases from bovine liver, yeast, and escherichia coli

Catalase is the enzyme which decomposes hydrogen peroxide to water and oxygen. Escherichia coli contains two catalases. Hydroperoxidase I (HPI) is a bifunctional catalase-peroxidase. Hydroperoxidase II (HPII) is only catalytically active toward H202. Expression of the genes encoding these proteins is controlled by different regimes. HPJI is thought to be a hexamer, having one heme d cis group per enzymatic subunit. HPII wild type protein and heme containing mutant proteins were obtained from the laboratory of P. Loewen (Univ. of Manitoba). Mutants constructed by oligonucleotidedirected mutagenesis were targeted for replacement of either the His128 residue or the Asn201 residue in the vicinity of the HPII heme crevice. His128 is the residue thought to be analogous to the His74 distal axial ligand of the heme in the bovine liver enzyme, and Asn201 is believed to be a residue critical to the function of the enzyme because of its role in orienting and interacting with the substrate molecule. Investigation of the nature of the hemes via absorption spectroscopy of the unmodified catalase proteins and their derived pyridine hemochromes showed that while the bovine and Saccharomyces cerevisiae catalase enzymes are protoheme-containing, the HPII wild type protein contains heme d, and the mutant proteins contain either solely protoheme, or heme d-protoheme mixtures. Cyanide binding studies supported this, as ligand binding was monophasic for the bovine, Saccharomyces cerevisiae, and wild type HPII enzymes, but biphasic for several of the HPII mutant proteins. Several mammalian catalases, and at least two prokaryotic catalases, are known to be NADPH binding. The function of this cofactor appears to be the prevention of inactivation of the enzyme, which occurs via formation of the inactive secondary catalase peroxide compound (compound II). No physiologically plausible scheme has yet been proposed for the NADPH mediation of catalase activity. This study has shown, via fluorescence and affinity chromatography techniques, that NADPH binds to the T (Typical) and A (Atypical) catalases of Saccharomyces cerevisiae, and that wild type HPII apparently does not bind NADPH. This study has also shown that NADPH is unlike any other hydrogen donor to catalase, and addresses its features as a unique donor by proposing a mechanism whereby NADPH is oxidized and catalase is protected from inactivation via the formation of protein radical species. Migration of this radical to a position close to the NADPH is also proposed as an adjunct hypothesis, based on similar electron migrations that are known to occur within metmyoglobin and cytochrome c peroxidase when reacted with H202. Validation of these hypotheses may be obtained in appropriate future experiments.

Fimbriae of coprinus cinereus, schizophyllum commune and ustilago violacea = structural aspects and a role in conjugation

Surface fibrils (fimbriae) have been observed on fungi from every major group. Fimbriae are thought to be involved in the following cell to cell interactions: conjugation, flocculation and adhesion. Several higher fungi exibit two other types of interactions: hyphal fusion (anastomosis) and clamp connection formation. As a prelude to examining the role of fimbriae in these processes, the fimbriae of two fungi that undergo these fusion events were examined. Electron microscopy studies revealed that Coprinus cinereus and Schizophyllum commune are fimbriated. C. cinereus fimbriae were 5 nm in diameter and 0.5 to 20 11m in length. Fimbriae of C. cinereus oidia were more numerous and longer than those of the hyphal stage. S. commune fimbriae were also 5 nm in diameter, but were only 0.5 to 2 11m in length. There was an unequal distribution of fimbriae on the hyphal surfaces of S. commune . Fimbriae were sparsely distributed over the entire hyphal surface, with higher densities of fibrils present on the side growths of the hyphae found in the older sections of the mycelium. Antiserum raised against Ustilago violacea fimbrial protein (AU) crossreacted strongly with 37 and 39 kd C. cinereus mycelial proteins. In contrast, AU bound very weakly to 89 and 92 kd S. commune mycelial proteins. Since AU cross-reacted poorly with S. commune fimbrial proteins, it was impossible to further characterize the fimbriae of this specIes. The 37 and 39 kd C. cinereus proteins, were isolated by electroelution and were shown to be able to form fibrils the same diameter as oidial fimbriae. The 37 kd protein was shown to be composed of several proteins with isoelectric points ranging from pH 6.1 to 7.63. Furthermore, the 37 kd protein was found to be multimeric, while the 39 kd protein was not. These results strongly suggested that the 37 kd protein is the structural fimbrial protein of C. cine reus . Finally, a series of experiments were designed to determine whether fimbriae are required for conjugation in U. violacea Conjugation was inhibited significantly with AU, but not with pre-immune serum or AU preincubated with purified fimbrial protein. Thus, it was concluded that fimbriae play a central role in mating in this organism.

Occupational therapist and client experiences of a mental health group

The purpose of this qualitative study was to understand the client and occupational therapist experiences of a mental health group. A secondary aim was to explore the extent to which this group seemed to have reflected a client-centred approach. The topic emerged from personal and professional issues related to the therapist as teacher and to inconsistencies in practice with the profession's client-centred philosophy. This philosophy, the study's frame of reference, was established in terms of themes related to the client-therapist relationship and to client values. Typical practice was illustrated through an extensive literature review. Structured didacticexperiential methods aiming toward skill development were predominant. The interpretive sciences and, to a lesser extent, the critical sciences directed the methodology. An ongoing support group at a community mental health clinic was selected as the focus of the study; the occupational therapist leader and three members became the key participants. A series of conversational interviews, the . core method of data collection, was supplemented by observation, document review, further interviews, and fieldnotes. Transcriptions of conversations were returned to participants for verification and for further reflection. Analysis primarily consisted of coding and organizing data according to emerging themes. The participants' experiences of group, presented as narrative stories within a group session vignette, were also returned to participants. There was a common understanding of the group's structure and the importance of having "air time" within the group; however, differences in perceptions of such things as the importance of the group in members' lives were noted. All members valued the therapeutic aspects of group, the role of group as weekly activity and, to a lesser extent, the learning that came from group. The researcher's perspective provided a critique of the group experience from a client-centred perspective. Some areas of consistency with client-centred practice were noted (e.g., therapist attitudes); however the group seemed to function far from a client-centred ideal. Members held little authority in a relationship dominated by the leaders, and leader agendas rather than member values controlled the session. Possible reasons for this discrepancy ranging from past health care encounters through to co-leader discord emerged. The actual and potential significance of this study was discussed according to many areas of implications: to OT practice, especially client-centred group practice, to theory development, to further areas of research and methodology considerations, to people involved in the group and to my personal growth and development.

An analysis of the systemic aspects of rights training for people with intellectual disabilities

Individuals with intellectual disabilities (ID) as a group have been subject to abuse. Individuals with ID need to be made aware of their rights. The 3Rs: Rights, Respect and Responsibility Human Rights Project is promoting rights awareness in individuals with ID, their caregivers and family members. To be effeCtive, abuse prevention must include support from the whole organization and its processes. This research evaluated the impact of the 3Rs initiative on the organization. It focused particularly on descriptions of organizational change perceived by full-time staff and managers in response to the initiation of the 3Rs Project. Behavioural interviews were conducted and a thematic analysis was used to describe changes in the organizational culture and behavioural mechanisms maintaining these changes. Systemic barriers to change were also explored. The results indicate that the Association is effectively implementing and supporting the rights-based philosophy.

Molybdenum (IV) imido silylamido and hydride complexes : stoichiometric and catalytic reactivity, mechanistic aspects of hydrosilation reactions

This thesis describes the synthesis, structural studies, and stoichiometric and catalytic reactivity of novel Mo(IV) imido silylamide (R'N)Mo(R2)(173_RIN-SiR32-H)(PMe3)n (1: Rl = tBu, Ar', Ar; R2 = Cl; R32 = Me2, MePh, MeCl, Ph2, HPh; n = 2; 2: R' = Ar, R2 = SiH2Ph, n = 1) and hydride complexes (ArN)Mo(H)(R)(PMe3)3 (R = Cl (3), SiH2Ph (4». Compounds of type 1 were generated from (R'N)Mo(PMe3)n(L) (5: R' = tBu, Ar', Ar; L = PMe3, r/- C2H4) and chlorohydrosilanes by the imido/silane coupling approach, recently discovered in our group. The mechanism of the reaction of 5 with HSiCh to give (ArN)MoClz(PMe3)3 (8) was studied by VT NMR, which revealed the intermediacy of (ArN)MCh(172 -ArN=SiHCl)(PMe3)z (9). The imido/silyl coupling methodology was transferred to the reactions of 5 with chlorine-free hydrosilanes. This approach allowed for the isolation of a novel ,B-agostic compound (ArN)Mo(SiHzPh)(173 -NAr-SiHPhH)(PMe3) (10). The latter was found to be active in a variety of hydrosilation processes, including the rare monoaddition of PhSiH3 to benzonitrile. Stoichiometric reactions of 11 with unsaturated compounds appear to proceed via the silanimine intermediate (ArN)M(17z-ArN=SiHPh)(PMe3) (12) and, in the case of olefins and nitriles, give products of Si-C coupling, such as (ArN)Mo(R)(173 -NAr-SiHPh-CH=CHR')(PMe3) (13: R = Et, R' = H; 14: R = H, R' = Ph) and (ArN)Mo(172-NAr-SiHPh-CHR=N)(PMe3) (15). Compound 13 was also subjected to catalysis showing much improved activity in the hydrosilation of carbonyls and alkenes. Hydride complexes 3 and 4 were prepared starting from (ArN)MoCh(PMe3)3 (8). Both hydride species catalyze a diversity of hydrosilation processes that proceed via initial substrate activation but not silane addition. The proposed mechanism is supported by stoichiometric reactions of 3 and 4, kinetic NMR studies, and DFf calculations for the hydrosilation of benzaldehyde and acetone mediated by 4.