Performance of Inaugural Student Cohorts in 1-Year Special Master's Program.

SUNY Upstate Medical University (Upstate) recently revised two 1-year special master's programs (SMPs), designed to enhance academics in preparation for medical school through rigorous graduate science coursework. Upstate SMP program success was measured by graduate performance during the first year of medical school. First-year performance measures included academic deficiencies, exam performance, and participation grades in a longitudinal case-based course on ethics and public health. Upstate SMP students with MCAT scores below the 50th percentile or who self-report classified as underrepresented in medicine outperformed controls on several metrics. Upstate SMP students consistently bested controls in the longitudinal course. Overall, Upstate SMP graduates performed commensurately with classmates.

Quick and Clean: LCME Scientific Method Training Without a Teaching Laboratory.

This exercise satisfies the Liaison Committee on Medical Education Standard 7.3 for medical student training in the scientific method. The students are challenged, individually and in small groups, to state and test hypotheses based on real patient data concerning risk factors for the development of hepatocellular carcinoma.

Formation of complex AChR aggregates in vitro requires alpha-dystrobrevin.

Efficient function at the neuromuscular junction requires high-density aggregates of acetylcholine receptors (AChRs) to be precisely aligned with the motor nerve terminal. A collaborative effort between the motor neuron and muscle intrinsic factors drives the formation and maintenance of these AChR aggregates. alpha-Dystrobrevin (alpha DB), a cytoplasmic protein found at the postsynaptic membrane, has been implicated in the regulation of AChR aggregate density and patterning. To investigate the contribution of alpha DB to the muscle intrinsic program regulating AChR aggregate development, we analyzed the formation of complex, pretzel-like AChR aggregates on primary muscle cell cultures derived from alpha DB knockout (alpha DB-KO) mice in the absence of nerve or agrin. In myotubes lacking alpha DB, complex AChR aggregates failed to form, whereas aggregates formed readily in wildtype myotubes. Five major isoforms of alpha DB are expressed in skeletal muscle: alpha DB1, alpha DB1(-), alpha DB2, alpha DB2(-), and alpha DB3. Expression of alpha DB1 or alpha DB1(-) in alpha DB-KO myotubes restored formation of complex AChR aggregates similar to those in wildtype myotubes. In contrast, individual expression of alpha DB2, alpha DB2(-), alpha DB3, or an alpha DB1 phosphorylation mutant resulted in the formation of few, if any, complex AChR aggregates. Collectively, these data suggest that alpha DB is a significant component of the muscle intrinsic program that mediates the formation of complex AChR aggregates and that alpha DB's tyrosine phosphorylation sites are of particular functional importance to this program. Although the muscle intrinsic program appears to influence synaptogenesis, the formation of complex mature AChR aggregates in alpha DB-KO mice (with the motor neuron present) suggests the motor neuron, not the muscle intrinsic program, is the major stimulus driving the maturation of AChRs from plaque to pretzel in vivo.

Mirk/dyrk1B is a Rho-induced kinase active in skeletal muscle differentiation.

The Rho family of small GTPases regulates numerous signaling pathways that control the organization of the cytoskeleton, transcription factor activity, and many aspects of the differentiation of skeletal myoblasts. We now demonstrate that the kinase Mirk (minibrain-related kinase)/dyrk1B is induced by members of the Rho-family in myoblasts and that Mirk is active in skeletal muscle differentiation. Mirk is an arginine-directed serine/threonine kinase which is expressed at elevated levels in skeletal muscle compared with other normal tissues. A Mirk promoter construct was activated when C2C12 myoblasts were switched from growth to differentiation medium and was also activated by the Rho family members RhoA, Cdc42, and to a lesser degree Rac1, but not by MyoD or Myf5. Mirk protein levels increased following transient expression of constitutively active Cdc42-QL, RhoA-QL, or Rac1-QL in C2C12 cells. High concentrations of serum mitogens down-regulated Mirk through activation of the Ras-MEK-Erk pathway. As a result, Mirk transcription was induced by the MEK1 inhibitor PD98059 and by the switch from growth to differentiation medium. Mirk was induced with similar kinetics to another Rho-induced differentiation gene, myogenin. Mirk protein levels increased 10-fold within 24-48 h after primary cultured muscle cells; C2C12 mouse myoblasts or L6 rat myoblasts were induced to differentiate. Thus Mirk was induced following the commitment stage of myogenesis. Stable overexpression of Mirk enabled myoblasts to fuse more rapidly when placed in differentiation medium. The function of Mirk in muscle differentiation was established by depletion of endogenous Mirk by small interfering RNA, which prevented myoblast fusion into myotubes and inhibited induction of markers of differentiation, including myogenin, fast twitch troponin T, and muscle myosin heavy chain. Other members of the dyrk/minibrain/HIPK family of kinases in lower organisms have been shown to regulate the transition from growth to differentiation, and Mirk is now shown to participate in skeletal muscle development.

Abnormal thalamocortical pathfinding and terminal arbors lead to enlarged barrels in neonatal GAP-43 heterozygous mice.

GAP-43 has been implicated in axonal pathfinding and sprouting, synaptic plasticity, and neurotransmitter release. However, its effect on cortical development in vivo is poorly understood. We have previously shown that GAP-43 knockout (-/-) mice fail to develop whisker-related barrels or an ordered whisker map in the cortex. Here we used cytochrome oxidase (CO) histochemistry to demonstrate that GAP-43 heterozygous (+/-) mice develop larger than normal barrels at postnatal day 7 (P7), despite normal body and brain weight. Using serotonin transporter (5HT-T) histochemistry to label thalamocortical afferents (TCAs), we found no obvious abnormalities in other somatosensory areas or primary visual cortex of GAP-43 (+/-) mice. However, TCA projections to (+/-) primary auditory cortex were not as clearly defined. To clarify the mechanism underlying the large-barrel phenotype, we used lipophilic (DiI) axon labeling. We found evidence for multiple pathfinding abnormalities among GAP-43 (+/-) TCAs. These axons show increased fasciculation within the internal capsule, as well as abnormal turning and branching in the subcortical white matter. These pathfinding errors most likely reflect failures of signal recognition and/or transduction by ingrowing TCAs. In addition, many DiI-labeled (+/-) TCAs exhibit widespread, sparsely branched terminal arbors in layer IV, reflecting the large-barrel phenotype. They also resemble those found in rat barrel cortex deprived of whisker inputs from birth, suggesting a failure of activity-dependent synaptogenesis and/or synaptic stabilization in (+/-) cortex. Our findings suggest that reduced GAP-43 expression can alter the fine-tuning of a cortical map through a combination of pathfinding and synaptic plasticity mechanisms.

Tyrosine-phosphorylated and nonphosphorylated isoforms of alpha-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions.

alpha-Dystrobrevin (DB), a cytoplasmic component of the dystrophin-glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking alphaDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main alphaDB isoforms, alphaDB1 and alphaDB2, with common NH2-terminal but distinct COOH-terminal domains. alphaDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. alphaDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in alphaDB-/- mice prevented muscle fiber degeneration; however, only alphaDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of alphaDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, alphaDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, alphaDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

Rapsyn-mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits.

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high-density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the alpha subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn-induced clustering of the AChR beta, gamma, and delta subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into alphadelta dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild-type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn-mediated clustering of an assembly intermediate, the alphadelta dimer.