Synaptogenesis at the neuromuscular junction requires agrin-induced stable localization of acetylcholine receptors (AChRs) at the endplate. The effects of agrin are transduced by the muscle-specific receptor tyrosine kinase (MuSK). This study provides evidence that Src-class protein tyrosine kinases mediate the effects of agrin-activated MuSK to regulate clustering and anchoring of AChRs in skeletal muscle. MuSK was complexed with both Src and Fyn in the C2 mouse muscle cell line. These associations were enhanced by agrin and by increasing protein tyrosine phosphorylation with pervanadate. Coupling between MuSK and the Src-class kinases in vivo appeared to be caused by a phosphotyrosine-SH2 domain interaction because binding of MuSK to the SH2 domains of Fyn and Src in vitro was specific, enhanced by phosphorylation, and dependent on MuSK autophosphorylation. In addition, Src and Fyn phosphorylated MuSK. AChR phosphorylation, stimulated by agrin or pervanadate, was inhibited by blocking Src-class kinases with PP1. Furthermore, agrin-induced clustering and cytoskeletal anchoring of AChRs was dependent on Src-family kinases. These data support the conclusion that Fyn and Src act downstream of MuSK to regulate the stable localization of AChRs at the neuromuscular endplate during agrin-induced synaptogenesis.

Background: Evaluation of newly diagnosed patients with melanoma for metastasis is requisite to treatment planning. The reported diagnostic yield of whole-body conventional radiological imaging in initial staging of patients with melanoma is low. However, the diagnostic yield of positron emission tomography (PET) for distant metastases is unclear. Hypothesis: There is no utility of PET as part of a routine metastatic survey in patients with T2 to T4 melanoma.

This study demonstrates that quantitative analysis from CT imaging is prognostic for control of the primary site when radiation therapy is given for treatment of supraglottic cancer.

In previous work (McKee EE, Bentley AT, Smith RM Jr, and Ciaccio CE, Biochem Biophys Res Commun 257: 466-472, 1999), the transport of guanine nucleotides into the matrix of intact isolated heart mitochondria was demonstrated. In this study, the time course and mechanisms of guanine nucleotide transport are characterized. Two distinct mechanisms of transport were found to be capable of moving guanine nucleotides across the inner membrane. The first carrier was saturable, displayed temperature dependence, preferred GDP to GTP, and did not transport GMP or IMP. When incubated in the absence of exogenous ATP, this carrier had a V(max) of 946 +/- 53 pmol. mg(-1). min(-1) with a K(m) of 2.9 +/- 0.3 mM for GDP. However, transport of GTP and GDP on this carrier was completely inhibited by physiological concentrations of ATP, suggesting that this carrier was not involved with guanine nucleotide transport in vivo. Because transport on this carrier was also inhibited by atractyloside, this carrier was consistent with the well-characterized ATP/ADP translocase. The second mechanism of guanine nucleotide uptake was insensitive to atractyloside, displayed temperature dependence, and was capable of transporting GMP, GDP, and GTP at approximately equal rates but did not transport IMP, guanine, or guanosine. GTP transport via this mechanism was slow, with a V(max) of 48.7 +/- 1.4 pmol. mg(-1). min(-1) and a K(m) = 4.4 +/- 0.4 mM. However, because the requirement for guanine nucleotide transport is low in nondividing tissues such as the heart, this transport process is nevertheless sufficient to account for the matrix uptake of guanine nucleotides and may represent the physiological mechanism of transport.