Ectopic expression of MITF, a gene for Waardenburg syndrome type 2, converts fibroblasts to cells with melanocyte characteristics.

MITF (microphthalmia-associated transcription factor) encodes a transcription factor with a basic-helix-loop-helix-zipper (bHLH-Zip) motif. MITF mutations occur in patients with Waardenburg syndrome type 2, a disorder associated with melanocyte abnormalities. Here we show that ectopic expression of MITF converts NIH/3T3 fibroblasts into cells with characteristics of melanocytes. MITF transfectants formed foci of morphologically altered cells, which resemble those induced by oncogenes, but did not exhibit malignant phenotypes. Instead, they contained dendritic cells that express melanogenic marker proteins such as tyrosinase and tyrosinase-related protein 1. Most cloned cells of MITF transfectants exhibited dendritic morphology and expressed melanogenic markers, but such properties were not observed in cells transfected with closely related TFE3 cDNA. Our findings indicate that MITF is critically involved in melanocyte differentiation.

Biological function of PDGF-induced PI-3 kinase activity: its role in alpha PDGF receptor-mediated mitogenic signaling.

The tyrosine phosphorylation sites in the human alpha PDGF receptor (alpha PDGFR) required for association with PI-3 kinase have been identified as tyrosines 731 and 742. Mutation of either tyrosine substantially reduced PDGF-induced PI-3 kinase activity but did not impair the receptor-mediated mitogenic response. We sought to determine whether PDGF-induced PI-3 kinase activity could be further ablated so as to exclude a low threshold requirement for PDGFR signal transduction. Thus, we mutated both tyrosine 731 and 742 and expressed the double mutant (Y731F/Y742F) in 32D hematopoietic cells. In such transfectants, PDGF induced no detectable receptor-associated or anti-P-Tyr recoverable PI-3 kinase activity. Under the same conditions, neither mobility shift of raf-1 nor tyrosine phosphorylation of either PLC gamma or MAP kinase was impaired. 32D transfectants expressing the double mutant showed wild-type alpha PDGFR levels of mitogenic and chemotactic responses to PDGF. To examine the effect of the double mutation in cells that normally respond to PDGF, we generated chimeras in which the cytoplasmic domains of wild-type alpha PDGFR, Y731F, and Y731F/Y742F were linked to the extracellular domain of colony-stimulating factor-1 (CSF-1) receptor (fms). After introduction of the chimeric receptors into mouse NIH/3T3 fibroblasts, the ability of CSF-1 to stimulate growth of these transfectants was examined. Our data show that all these chimeric receptors exhibited similar abilities to mediate CSF-1-stimulated cell growth. These findings lead us to conclude that PDGF-induced PI-3 kinase activity is not required for PDGF-stimulated mitogenic pathway in both NIH/3T3 fibroblasts and 32D hematopoietic cells.

Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells.

Nerve growth factor induces differentiation and survival of rat PC12 pheochromocytoma cells. The activation of the erk cascade has been implicated in transducing the multitude of signals induced by NGF. In order to explore the role of this signaling cascade in NGF mediated survival, differentiation and proliferation, we generated recombinant adenoviruses which express the intermediates of the erk cascade in their wild type, dominant negative and constitutively activated forms. We show that differentiation of PC12 cells requires activity of the ras/erk pathway, whereas inhibition of this pathway had no effect on survival or proliferation. Constitutively active forms of ras, raf and mek induced PC12 cell differentiation, while dominant interfering forms inhibited differentiation. Survival of PC12 cells in serum-free medium did not require activity of the ras/erk pathway. Instead, PI3 Kinase signaling was necessary for PC12 cell survival. Interestingly, constitutively activated versions of raf and mek were able to promote survival, but again this was dependent on activation of PI3 Kinase. Therefore, at least two distinct signaling pathways are required in PC12 cells for mediation of NGF functions.

Disseminated gonococcal infection in elderly patients.

Four elderly patients (71, 53, 57, and 62 years old) had disseminated gonococcal infection. Three patients presented with suppurative arthritis and the fourth with fever, skin lesions, and malaise. Although the signs and symptoms did not differ from those in the younger age group, the diagnosis was not considered clinically. All gonococci were susceptible to penicillin.

Adoptive transfer of experimental allergic encephalomyelitis using serum-free, chemically defined in vitro culture conditions.

Enhanced adoptive transfer of experimental allergic encephalomyelitis (EAE) in rats requires in vitro culture of encephalitogen-sensitized donor spleen cells with either myelin basic protein or T-cell mitogen for 18-72 h prior to transfer to unimmunized recipients. The required in vitro culture period offers an opportunity to address in detail cellular and molecular immunoregulatory processes involved in the development of EAE. Conventional culture conditions using fetal bovine serum may impose analytical limitations due to the chemical complexity of the media. To permit better definition of the chemical events associated with the development of EAE, we report the successful adoptive transfer of EAE in Lewis rats using completely chemically defined, serum-free culture conditions.

Traditional and phonological treatment for teaching English fricatives and affricates to Koreans.

Non-native speakers of English are being seen for accent reduction in speech-language pathology clinics although there is little evidence that treatment is effective. Two types of treatment were evaluated. Traditional articulation-based treatment was successful for 2 native Korean speakers in improving English fricatives and affricate production. A second treatment using phonologically based contrasts was also successful for the same sounds with 2 other Korean speakers. Some issues in foreign accent reduction are discussed.

A human oncogene of the RAS superfamily unmasked by expression cDNA cloning.

As an approach to identify human oncogenes, we generated an expression cDNA library from an ovarian carcinoma line. A potent transforming gene was detected by transfection analysis and identified as TC21, a recently cloned member of the RAS gene superfamily. A single point mutation substituting glutamine for leucine at position 72 was shown to be responsible for activation of transforming properties. While the cDNA clone possessed high transforming activity, the ovarian tumor genomic DNA, which contained the mutated TC21 allele, failed to induce transformed foci. Thus, expression cDNA cloning made it possible to identify and isolate a human oncogene that has evaded detection by conventional approaches.