Robert J. Milner

Identifying the protein products of brain-specific genes with antibodies to chemically synthesized peptides

From the nucleotide sequences of three cDNA clones of rat brain-specific mRNAs, we deduced the partial amino acid sequences of two previously unknown proteins. We raised antisera to synthetic peptides mimicking short regions of these putative brain-specific proteins, and used these sera in immunocytochemical studies to localize each protein in the brain. One protein is found in large neurons throughout the brain, asymmetrically distributed toward the dendritic pole of the cell cytoplasm, suggesting involvement in the synthesis and/or directional transport of dendritic substances. The sequence of the second protein contains pairs of basic residues similar to precursors for neurotransmitters. The protein is located in (and may be the precursor for the neurotransmitter of) a novel fiber network of major extent with ramifications in cerebellum, hippocampus, and cortex, and in cell bodies in the brain stem, hypothalamus, and caudate nucleus. Our approach provides a direct method for characterizing rare brain molecules which may not have been anticipated and for which there are no known functional assays.

A Single Nucleotide Difference in the Gene for Myelin Proteolipid Protein Defines the Jimpy Mutation in Mouse

We have previously shown that, in the myelin‐deficient jimpy mutant mouse, 74 nucleotides are absent from the mRNA for proteolipid protein (PLP) as a result of aberrant RNA processing. To define the exact site of the jimpy mutation, we have analyzed the PLP gene obtained from a jimpy mouse genomic library. We find that the nucleotide sequence that is absent from jimpy PLP mRNA is fully preserved in the jimpy PLP gene. The missing segment corresponds to a separate exon, equivalent to exon 5 of the human PLP gene. The nucleotide sequence at the 3’end of intron 4 in the jimpy PLP gene contains a single point mutation. A base change A → G in the 3’acceptor splice site has altered a position that is 100% conserved in all published splice acceptor sequences. We conclude that the primary genetic defect of the jimpy mouse is a single base change in the PLP gene disabling an invariant recognition sequence of RNA splicing.

Functional Mentoring: A Practical Approach With Multilevel Outcomes

Introduction: Mentoring is a central component of professional development. Evaluation of “successful” mentoring programs, however, has been limited and mainly focused on measures of satisfaction with the relationship. In todays environment, mentoring programs must produce tangible outcomes to demonstrate success. To address this issue, the authors advance the framework of functional mentoring combined with measurement of outcomes at multiple levels. Methods: The mentoring program is embedded within an intensive, continuing medical education (CME) accredited faculty development program. Survey methodology is used to collect qualitative and quantitative data at the start, midpoint, and end of the program and longitudinally. Participants in 4 years of the program were surveyed. Results: In 4 years, 165 faculty participated in the program. Respondents were highly satisfied with the pairings: 85% of junior faculty believed their mentor had a significant effect on their projects. Junior faculty reported a significant enhancement of skills related to initiating and negotiating a new mentoring relationship (85%) and stated that their project would have a significant impact on their career (92%) and on the department or institution (86%). Discussion: The success of this mentoring program is demonstrated at multiple levels. The key outcome of functional mentoring is the project. Projects are aligned with professional responsibilities and with institutional missions. The project contributes to the individuals dossier and adds value to the institution. Functional mentoring is a practical approach that allows measurable results at multiple levels.

Neural Protein 1B236/Myelin-Associated Glycoprotein (MAG) Defines a Subgroup of the Immunoglobulin Superfamily

We have reviewed the structure and properties of the neural protein 1B236/MAG. This molecule consists largely of five Ig-like domains separated from its carboxyl terminal tail by a single membrane-spanning region. Two forms of the protein differ in the length and sequence of the carboxyl terminus: these are encoded by alternatively spliced mRNAs that are differentially expressed during postnatal neural development. The Ig-like domains of 1B236/MAG are unusual in having structural similarities to Ig V domains but with short Cys-Cys distances characteristic of C domains. Several other Ig-like molecules exhibit this structural feature, including the cell adhesion molecule N-CAM, which is most closely related in sequence to 1B236/MAG. We have proposed 1B236/MAG as the prototype for this subgroup of the Ig family and offer a model for this type of Ig domain structure. 1B236/MAG probably acts as a cell adhesion molecule to mediate interactions between cells in a fashion similar to that proposed for N-CAM. In particular, 1B236/MAG may be involved in interactions between myelinating oligodendrocytes or Schwann cells and axons or between adjacent layers of myelin membrane during the process of myelin compaction. It is most likely that the homophilic or heterophilic interactions of 1B236/MAG occur through binding to the Ig-like domains. The structure of 1B236/MAG is therefore quite consistent with its proposed function and may serve as the model for this class of cell-cell interaction molecules. One would predict, for example, that the neuron-glia cell adhesion molecule Ng-CAM, also known as NILE or L1 (Bock et al. 1985, Friedlander et al. 1985), which mediates interactions between neurons and glial cells, would have a very similar structure to those of N-CAM and 1B236/MAG. In addition, the carboxyl terminal tails of the 1B236/MAG proteins may also be involved in interactions with cytoskeletal components, during membrane vesicle transport through the glial cytoplasm during myelination or through neuronal axoplasm or cytoplasm. The availability of full-length cDNA clones of 1B236/MAG mRNAs with the ability to express these products in vitro will enable the structure and interactions of 1B236/MAG to be tested in detail.

The essential value of projects in faculty development.

Projects--planned activities with specific goals and outcomes--have been used in faculty development programs to enhance participant learning and development. Projects have been employed most extensively in programs designed to develop faculty as educators. The authors review the literature and report the results of their 2008 study of the impact of projects within the Pennsylvania State University College of Medicine Junior Faculty Development Program, a comprehensive faculty development program. Using a mixed-methods approach, the products of project work, the academic productivity of program graduates, and the impact of projects on career development were analyzed. Faculty who achieved the most progress on their projects reported the highest number of academic products related to their project and the highest number of overall academic achievements. Faculty perceived that their project had three major effects on their professional development: production of a tangible outcome, development of a career focus, and development of relationships with mentors and peers. On the basis of these findings and a review of the literature, the authors conclude that projects are an essential element of a faculty development program. Projects provide a foundation for future academic success by enabling junior faculty to develop and hone knowledge and skills, identify a career focus and gain recognition within their community, generate scholarship, allocate time to academic work, and establish supportive relationships and collaborative networks. A list of best practices to successfully incorporate projects within faculty development programs is provided.

Detection of the messenger RNA coding for preproenkephalin A in bovine adrenal by in situ hybridization

The messenger RNA (mRNA) coding for the adrenal precursor of enkephalins (preproenkephalin-A) has been detected in bovine adrenal medulla cells using in situ hybridization with 32P-labelled preproenkephalin A (PPA) complementary DNA. In formaldehyde- and Carnoy-fixed tissue sections, an intense elective labelling restricted to the cells located at the periphery of the adrenal medulla can be detected after hybridization procedure, using X-ray film and classical autoradiographic procedure. Adequate controls show that this labelling is obtained only using PPA complementary DNA, inserted or not in its vector. Distribution of PPA mRNA appears identical to that of its immunoreactive end products, namely Met-enkephalin and BAM22 peptide, detected by immunohistochemistry. Norepinephrine, detectable using monoamine histofluorescence, appears restricted to the cells of the center of the gland unlabelled for PPA mRNA and its end-products. Cultured bovine adrenomedullary cells that exhibited enkephalin immunoreactivity also contain PPA mRNA located in their cytoplasm.

Differential expression of protein tyrosine kinase genes during microglial activation

Protein tyrosine kinase (PTK) activity is abundant in microglia, but the PTKs that participate in their activation have not been identified. For these studies, we used three paradigms to characterize PTK expression during microglial activation: resting and activated microglia were bulk fractionated from the adult brain, cultured newborn microglia were treated with lipopolysaccharide (LPS) to model the transition from activated toward phagocytic microglia, and PTK expression was examined in activated microglia in situ after facial nerve axotomy. Two PCR‐based strategies were used to show that 21 different PTK genes are expressed by rat brain microglia: 5 receptor PTKs, 10 nonreceptor PTKs, and 6 members of the src family. Seven of the 21 PTKs were examined in greater detail. Five PTK mRNAs (fgr, hck, fak, jak‐2, and flk‐1) increased expression across all three models of activation. We conclude that they represent key components in the cascades that participate in microglial activation. In contrast, expression of fes and fms correlated with stimuli that affect microglial proliferation. Four of the PTKs (hck, fgr, fes, and fms) are believed to be myeloid cell specific and were not expressed by cultured astrocytes. HCK and FAK protein were also not expressed in lysates of immature astrocytes and oligodendrocytes. Because of their putative specificity, these kinases represent potential targets for inhibitors of microglial activation. Because reactive microglia can exacerbate the severity of neurological diseases, the identification of specific kinases that participate in microglial activation represents an important advance toward the development of new therapeutics. GLIA 40:11–24, 2002.

Perspective: Toward a competency framework for faculty.

Today, faculty in academic medicine face challenges in all three mission areas--research, education, and patient care--and require a broad set of competencies to survive in this changing environment. To support faculty and to design assessments that match new expectations, the authors argue that it is essential to capture the full scope of skills, knowledge, and behaviors necessary for a successful faculty member. Thus, it is timely to explore and define competencies for faculty in academic medicine. The authors describe three approaches to identifying faculty competencies. Each reveals diverse but overlapping sets of competency domains, reflecting the breadth of activities expected of todays faculty. To organize these competencies into a coherent framework, the authors propose a model based on a typology of competency. A key feature of the model is the division between occupational competencies, which are largely role-specific, and personal competencies, which are necessary for all faculty. A competency framework also must be developmental, to reflect the growth in skills, knowledge, and behaviors from trainee to expert and to allow for an individuals changing roles over a career. Such a competency framework will inform professional development activities and require assessment of competence. The generation of competencies also will reveal areas of faculty practice that are poorly measured, requiring new tools to be incorporated into existing processes of faculty evaluation. The authors provide general principles to guide the identification of a competency framework for faculty and invite the academic medicine community to engage in further discussion.

Alternative mRNA splicing: the Shaker gene

*Department of Molecular Biolo~ and tDivision of Preclinical Neuroscience and Endocrinolo~, Research Institute of SC~S Clinic, 106r;6 N. Torrey Pines Road La ]olla, CA 92037, USA. One of the surprising findings from the past decade of molecular biology researchis that a single gene can give rise to more than one mature mRNA via the process of alternative mRNA splicing. This phenomenon occurs in a substantial proportion of genes: for example, of known genes expressed predominantly in the nervous system, some 30% utilize alternative splicing. Most alternative splicing events af- fect the region of the mRNA that encodes protein, thus one biolo~cal rationale for this process is apparent: the alternative mRNAs encode pro- teins that share many characteristics with each other but differ in those properties harbored by the alter- natively ceded regions. Thus, the alternative protein products of a gene represent a protein family. Potassium channels The functional significance of al- ternative splicing is made extremely evident in molecular studies of voltage-gated potassium channels. Action potentials are initiated by depolarizations of the axon mem- brane that activate an inward Na + conductance. Action potentials are self-limiting in that, as membranes become depolarized, the conduc- tance to outward flow of K + also increases, thereby restoring, after 0.2-0.5ms, the membrane potential and the resting ,;alue of the Na + conductance. Several K + channels mediate membrane repolarization and these have been found to vary in their electrophysiological proper- ties, specifically in the rates at which they cause repolarization. Because the duration of depolarization at nerve terminals determines the amount of transmitter released, the type of K + channel expressed at a particular terminal may specify the strength of its synaptic interaction. To understand the variation in the electrophysiological properties of K + channels, it was essential to under- stand the biochemical nature of the different channel types. However, at the time there was neither antibody nor protein sequence information available to use in the construction of molec-l~r probes for cDNA doning. Instead, the characterization of K + channels was initiated by analysis of the Shaker (Sh) gene of Drosophila ~hu~aster and offers an example of how one may proceed from a gene to a description of its products. Sbakerflies Drosophila Sb mutants were initi- ally identified because, after ether anesthesia, the legs of mutant flies shook. The mutants exhibit an al- tered K + current identified as the A- current (Ia). The altered current was associated with prolonged action potentials and increased neurotrans-

Brain-specific gene expression.

The brain of an adult rat expresses approximately 30,000 different brain-specific mRNAs. To investigate their encoded proteins, we have selected cDNA clones corresponding to mRNAs expressed exclusively in rat brain, determined their nucleotide sequences and generated antisera against synthetic peptides mimicking short regions of the deduced protein sequences. The clone plB236 encodes a protein that defines a widely distributed neuronal system and may be the precursor for a family of novel neuropeptides. A second clone, plB208, encodes rat brain proteolipid protein, the major protein component of central nervous system myelin. These studies have also identified an 82 nucleotide genetic element called an ID (identifier) sequence that may be involved in the regulation of transcription of brain-specific genes.