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Donal A Walsh

Publications and source records attributed to Donal A Walsh.

9 recordsLinked to original sources
PubMed2004

Baculovirus-mediated overexpression of the phosphorylase b kinase holoenzyme and alpha gamma delta and gamma delta subcomplexes.

Recombinant baculoviruses were created and used to coexpress rat phosphorylase kinase (Phk) alpha, gamma, and delta subunits and rabbit beta subunit in insect cells. Coexpression allowed creation of the (alphabetagammadelta)4 hexadecamer, the alphagammadelta heterotrimer, and the gammadelta heterodimeric subcomplexes. Neither the individual alpha, beta, or gamma subunit nor any complex containing the beta subunit other than the hexadecameric holoenzyme was obtained in soluble form. The expressed complexes exhibited pH- and [Ca2+]-dependent specific activities that were similar to those of the Phk holoenzyme purified from rabbit skeletal muscle (SkM Phk). SkM Phk, expressed Phk, and the alphagammadelta subcomplex were activated by exogenous calmodulin and underwent Ca(2+)-dependent autophosphorylation. In some of these features there were subtle differences that could likely be attributed to differences in the covalent modification state of the baculovirus-driven expressed protein. Our results provide an important avenue to probe the detailed characterization of the structure of Phk and the function of the individual domains of the subunits using baculovirus-mediated expression of Phk and Phk subcomplexes.

Animals↗
PubMed2002

A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

Alternative Splicing↗
PubMed2002

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase.

The amount of phosphorylase kinase in skeletal muscle is exquisitely sensitive to developmental signals such as differentiation and innervation, and is clearly regulated in such a manner so as to always maintain the gamma catalytic subunit under the control of its regulatory alpha, beta and gamma subunits. To identify how the transcription of the gamma subunit is regulated, we have analysed 3.8 kb of the upstream regulatory region using a luciferase reporter system. A complex sequence of interdependent regulations is evident. The gamma catalytic subunit gene contains two inhibitory controls with very dominant features. Also evident are an array of multiple positive regulatory elements, prominent amongst which are four E-boxes, of which two are downstream, one is upstream and one is in the middle of the CAAT-TATA core promoter. Differentiation-dependent positive regulation arises as a consequence of both E-box regulation and the activation of at least one other regulatory element. The primary mode of transcriptional regulation of the gamma catalytic subunit gene appears to occur by the relief of regulation of an otherwise default inhibitory status. It is noteworthy that such a mode of regulation mirrors the regulation of the enzymic activity of many protein kinases, including phosphorylase kinase. With phosphorylase kinase, both its transcriptional regulation as well as the regulation of the protein itself, are primed to maintain the gamma catalytic subunit either unexpressed or inactivate respectively, until a positive signal occurs to override an otherwise dominant default inhibitory condition.

Animals↗
PubMed2002

A structural model of the catalytic subunit-regulatory subunit dimeric complex of the cAMP-dependent protein kinase.

Previous neutron scattering studies elaborated the topographical relationship of the regulatory (R(IIalpha)) and catalytic (C(alpha)) subunits of the cAMP-dependent protein kinase. We present here the results of a set of computations that lead to an atomic model of the cAMP-dependent protein kinase heterodimer, Delta(1-91)R(IIalpha)-C(alpha). The first step in the modeling utilized the crystal structures for the porcine C(alpha) and bovine Delta(1-90)R(Ialpha) or rat Delta(1-111)R(IIbeta), to homology-model structures of the species and isoforms that had been used in the neutron scattering experiments (bovine C(alpha) subunit and murine Delta(1-91)R(IIalpha) subunit, respectively). A docking procedure, constrained by the dimensions and positions of the ellipsoids in the neutron-derived R-C model as well as mutagenesis data, was used to develop "best fit" models for the heterodimer. Simulated annealing, molecular dynamics, and energy minimization were then used to refine the side chain packing at the heterodimer interface. For comparison, the calculations were done using the homology models derived from both the R(Ialpha) and R(IIbeta) crystal structures. Both resultant models had many similarities. Each predicted similar interfaces. The R(Ialpha)-based model has 25% more hydrogen bonds than that based on R(IIbeta), with seven of these potential bonds in common. The distribution of hydrophobic, polar, and charged residues at the interface was similar for both models, with a distribution more characteristic of the exposed surface residues than those in the protein interior. The calculated interface area in each is relatively small (<2000 A(2)). The R(Ialpha)-based model, however, has a significantly better fit with the scattering data and is therefore the one of distinctly higher probability. With its small interface area that has a high proportion of charged and polar residues, the complex appears poised for dissociation, and each subunit existing as a stable entity. This result is consistent with the known physiological events required for cAMP-dependent activation of the kinase.

Amino Acid Sequence↗
PubMed2002

Defining the attributes expected of graduating veterinary medical students, part 2: external evaluation and outcomes assessment.

We have previously defined a set of 62 attributes-12 in the area of professional characteristics, 28 addressing knowledge and understanding, and 22 delineating skills-that veterinary students should be expected to have demonstrated by the time of their graduation (Walsh DA, Osburn BI, Christopher MM. Defining the attributes expected of graduating veterinary medical students. J Am Vet Med Assoc 219:1358-1365, 2001). We have used this set of attributes as the basis of an outcomes assessment completed by California practitioners to determine whether graduates from the University of California School of Veterinary Medicine are meeting these expectations. Based upon this assessment, these 62 defined attributes appear to reflect very well practicing veterinarians' views and expectations of DVM graduates. The survey results also indicate that, overall, the recent University of California graduates are meeting these set of expectations. Simultaneously, the outcomes assessment focused attention on several areas, including private practice management, work expectations for successful practice, and surgical capabilities. For each, California practitioners recommended that the definition of the expectation be expanded and that the level of achievement by graduates be improved. Defining a set of attributes expected of veterinary graduates is a key step in obtaining an effective outcomes assessment of a professional educational program.

Animals↗
PubMed2002

Template for a recommended curriculum in "Veterinary Professional Development and Career Success".

Recent studies of the veterinary profession have established a need for training in various areas beyond those directed toward building competence and proficiency as a clinician. To address this need, a workshop was designed whose objective was to develop a detailed outline of a model curriculum that would encompass the skills, knowledge, aptitudes, and attitudes deemed essential for economic success in the veterinary profession. The model curriculum was created from comprehensive input provided by consultants and educators. Constraints for implementation of this curriculum are identified, and future directions are discussed.

Curriculum↗