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Biomedical subjects

K R MacKenzie

Publications and source records attributed to K R MacKenzie.

At least 19 recordsLinked to original sources
PubMed2001

Interpersonal psychotherapy group (IPT-G) for depression.

A case study of a time-limited interpersonal psychotherapy group (IPT-G) is presented to illustrate the use of interpersonal therapy (IPT) to treat patients with major depression in a group psychotherapy format. The use of individual outcome measures as a helpful adjunct to clinical psychotherapeutic practice is demonstrated. Because IPT-G has only a few exclusion criteria (active suicidality and significant borderline personality features), it can be used in a broad range of clinical settings. This clinical example demonstrates IPT-G to be a useful modality for addressing a common and difficult patient population.

Adult↗
PubMed2000

Modulation of glycophorin A transmembrane helix interactions by lipid bilayers: molecular dynamics calculations.

Starting from the glycophorin A dimer structure determined by NMR, we performed simulations of both dimer and monomer forms in explicit lipid bilayers with constant normal pressure, lateral area, and temperature using the CHARMM potential. Analysis of the trajectories in four different lipids reveals how lipid chain length and saturation modulate the structural and energetic properties of transmembrane helices. Helix tilt, helix-helix crossing angle, and helix accessible volume depend on lipid type in a manner consistent with hydrophobic matching concepts: the most relevant lipid property appears to be the bilayer thickness. Although the net helix-helix interaction enthalpy is strongly attractive, analysis of residue-residue interactions reveals significant unfavorable electrostatic repulsion between interfacial glycine residues previously shown to be critical for dimerization. Peptide volume is nearly conserved upon dimerization in all lipid types, indicating that the monomeric helices pack equally well with lipid as dimer helices do with one another. Enthalpy calculations indicate that the helix-environment interaction energy is lower in the dimer than in the monomer form, when solvated by unsaturated lipids. In all lipid environments there is a marked preference for lipids to interact with peptide predominantly through one rather than both acyl chains. Although our trajectories are not long enough to allow a full thermodynamic treatment, these results demonstrate that molecular dynamics simulations are a powerful method for investigating the protein-protein, protein-lipid, and lipid-lipid interactions that determine the structure, stability and dynamics of transmembrane alpha-helices in membranes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Structure-based prediction of the stability of transmembrane helix-helix interactions: the sequence dependence of glycophorin A dimerization.

The ability to predict the effects of point mutations on the interaction of alpha-helices within membranes would represent a significant step toward understanding the folding and stability of membrane proteins. We use structure-based empirical parameters representing steric clashes, favorable van der Waals interactions, and restrictions of side-chain rotamer freedom to explain the relative dimerization propensities of 105 hydrophobic single-point mutants of the glycophorin A (GpA) transmembrane domain. Although the structure at the dimer interface is critical to our model, changes in side-chain hydrophobicity are uncorrelated with dimer stability, indicating that the hydrophobic effect does not influence transmembrane helix-helix association. Our model provides insights into the compensatory effects of multiple mutations and shows that helix-helix interactions dominate the formation of specific structures.

Amino Acid Sequence↗
PubMed1997

A transmembrane helix dimer: structure and implications.

The three-dimensional structure of the dimeric transmembrane domain of glycophorin A (GpA) was determined by solution nuclear magnetic resonance spectroscopy of a 40-residue peptide solubilized in aqueous detergent micelles. The GpA membrane-spanning alpha helices cross at an angle of -40 degrees and form a small but well-packed interface that lacks intermonomer hydrogen bonds. The structure provides an explanation for the previously characterized sequence dependence of GpA dimerization and demonstrates that van der Waals interactions alone can mediate stable and specific associations between transmembrane helices.

Amino Acid Sequence↗
PubMed1996

Leucine side-chain rotamers in a glycophorin A transmembrane peptide as revealed by three-bond carbon-carbon couplings and 13C chemical shifts.

We have used a spin-echo difference NMR pulse sequence to measure three-bond J couplings between lambda- and alpha-carbons of the leucine residues in a micelle-associated helical peptide dimer that corresponds to residues 62-101 of the transmembrane erythrocyte protein glycophorin A. The observed 3J couplings correlate strongly with the 13C chemical shift of the lambda-methyl groups, and within experimental error both the shift distribution of the methyl carbons and the variations in 3J can be accounted for by variations in side-chain rotamer populations. We infer that all leucine side chains in this peptide dimer are in fast exchange among chi 2 rotamers and sample two of the three possible rotameric states, even when the side chain forms part of the dimer interface. The observed correlation of chemical shift with couplings can be traced to a gamma-gauche interaction of methyl and alpha-carbons. This correlation may provide an alternate route to rotamer analysis in some protein systems.

Carbon Isotopes↗
PubMed1996

Mapping the lipid-exposed surfaces of membrane proteins.

Phospholamban forms a stable complex of five long transmembrane helices. We show that the relative rotational orientation of the helices in the pentameric complex can be distinguished by S-H to S-D exchange of cysteine sulphydryl groups located in the transmembrane segment of the protein and exposed to the lipid environment. Of the three cysteine residues in phospholamban, two residues (Cys 36 and Cys 46) are oriented towards the helix interface and protected from exchange, while the third cysteine (Cys 41) is oriented towards the lipid interface and undergoes exchange with water diffused into the bilayer. Distinguishing the external and internal faces of a membrane protein by sulphydryl exchange provides a general approach for determining the three-dimensional fold of membrane proteins and enhances model building efforts to generate high-resolution structures.

Adenosine Triphosphatases↗
PubMed1996

Time-limited group psychotherapy.

The presence of a time limit shapes the entire therapeutic process and culminates in the management of termination. This paper outlines a protocol for dealing with termination in time-limited psychotherapy groups: (1) Select suitable members with similar capacity to use a time-limited format; (2) use a closed group format; (3) clarify the time boundaries at the beginning; (4) reinforce the time frame during the last half of the group; (5) provide focused interventions; (6) forestall premature termination; (7) reinforce termination themes of deprivation, resentment and anger, rejection, grief and loss, responsibility for self; (8) structure the final session; and (9) plan a 4-month follow-up visit to encourage ongoing application. These guidelines provide the therapist with a general structure to ensure that major termination themes are systematically addressed. They deal with powerful concepts of maturation and self-responsibility and incorporate the principal strategies that distinguish time-limited from time-unlimited group psychotherapy.

Humans↗
PubMed1995

Overexpression, purification, and characterization of Escherichia coli acyl carrier protein and two mutant proteins.

A synthetic gene of 237 bases encoding the 77-residue acyl carrier protein (ACP) from Escherichia coli, along with two mutant genes, ACP-I54V and ACP-A59V, were subcloned into the pET11a-pLysS E. coli overexpression system under the control of the bacteriophage T7 promoter. This efficient expression system and a simplified purification protocol yielded more than 120 mg/l of pure protein. The construct produced a mixture of holo-ACP and apo-ACP and two HPLC procedures were developed to separate the two species. This overexpression system allows cost-effective growths of 13C- and 15N-labeled protein for structural and other studies on ACP. In the course of the work on the mutants of ACP, an apparent homologous recombination event led, in one case, to reversion to a wild-type protein, suggesting that precautions to prevent such reversion should be taken.

Acyl Carrier Protein↗
PubMed1994

Structural organization of the pentameric transmembrane alpha-helices of phospholamban, a cardiac ion channel.

Phospholamban is a 52 amino acid calcium regulatory protein found as pentamers in cardiac SR membranes. The pentamers form through interactions between its transmembrane domains, and are stable in SDS. We have employed a saturation mutagenesis approach to study the detailed interactions between the transmembrane segments, using a chimeric protein construct in which staphylococcal nuclease (a monomeric soluble protein) is fused to the N-terminus of phospholamban. The chimera forms pentamers observable in SDS-PAGE, allowing the effects of mutations upon the oligomeric association to be determined by electrophoresis. The disruptive effects of amino acid substitutions in the transmembrane domain were classified as sensitive, moderately sensitive or insensitive. Residues of the same class lined up on faces of a 3.5 amino acids/turn helical projection, allowing the construction of a model of the interacting surfaces in which the helices are associated in a left-handed pentameric coiled-coil configuration. Molecular modeling simulations (to be described elsewhere in detail) confirm that the helices readily form a left-handed coiled-coil helical bundle and have yielded molecular models for the interacting surfaces, the best of which is identical to that predicted by the mutagenesis. Residues lining the pore show considerable structural sensitivity to mutation, indicating that care must be taken in interpreting the results of mutagenesis studies of channels. The cylindrical ion pore (minimal diameter of 2 A) appears to be defined largely by hydrophobic residues (I40, L43 and I47) with only two mildly polar elements contributed by sulfurs in residues C36 and M50.

Amino Acid Sequence↗
PubMed1994

Where is here and when is now? The adaptational challenge of mental health reform for group psychotherapy.

The structure of health care delivery is in a period of rapid change. An understanding of psychotherapy service use patterns indicates the importance of time categories as well as the relevance of group techniques. Traditional interpersonal and psychodynamic group techniques may be modified for time-limited use without sacrificing basic values and with demonstrated effectiveness. Adapting to a more fiscally stringent practice environment requires the clinician to undertake a difficult process of transition. As professionals our task is to prepare ourselves for this challenge and to let the health care system know that proper practice guidelines are necessary to achieve effective treatment for our patients.

Cost Control↗
PubMed1988

Recent developments in brief psychotherapy.

The clinical effectiveness and many of the therapeutic mechanisms of brief psychotherapy have been validated through research studies in recent years, as indicated in this review of developments in the psychodynamic and interpersonal brief therapies. However, the literature shows considerable variation in methods for establishing time limits and in exclusion criteria. In a major shift in therapeutic practice, interpersonal rather than intrapsychic concepts are used to identify the focus of therapy, and systematic techniques for defining the therapeutic focus have been developed. The therapeutic alliance and its relation to outcome also have received substantial attention. Trends in service delivery clearly indicate increasing emphasis on brief psychotherapy, which must be conceptualized as a specific modality, not just as long-term therapy compressed.

Humans↗