Faculty Bibliography

Several treatments which regulate tyrosine hydroxylase (TH) transcription, such as stress in vivo, or 12-O-tetradecanoylphorbol-13-acetate (TPA) in cell culture, induce both Egr1 and AP1 factors. Previously, we identified a functional Egr1 motif overlapping with Sp1 site in the rat TH promoter. Its response to Egr1 also required the presence of an AP1/Ebox motif. Here, we further examined the cross-talk between these sites. Insertion of 10- or 20-bp between the Sp1/Egr1 and AP1/Ebox elements, reduced the ability of Egr1 to upregulate luciferase reporter activity controlled by the proximal 272 nucleotides of the rat TH promoter in PC12 cells. Electrophoretic mobility shift assays with nuclear extracts from TPA treated cells were used to identify the composition of the factors which bound the AP1/Ebox motif and whether there is competition with factors which bind the Sp1/Egr1 motif. The complexes formed with labeled AP1/E box oligonucleotide were reduced or supershifted with antisera to Fos family, c-Fos, Fra-2, and Jun D. Excess Sp1/Egr1 oligonucleotide or anti Egr1 antisera did not compete. Fra-2 was a major component of the complex after 2-4 h TPA. Transfection of PC12 cells with Fra-2 induced reporter activity requiring the AP1, but not the Egr1 motif. However, when cotransfected with Fra-2, Egr1 expression plasmids elicited lower induction of luciferase activity than observed with Egr1 alone. Our results suggest that although it does not compete for binding to the promoter, Egr1 can modulate the regulation of TH transcription by AP1 factors.

Latent transforming growth factor binding proteins (Ltbp-1, -2, -3 and -4) and fibrillins (Fbn-1 and -2) are structurally related cysteine-rich extracellular matrix proteins that localize to the 10 nm microfibrils. Ltbp-1 is thought to promote the secretion and proper folding of the small latent transforming growth factor beta (TGF-beta) complex (TGF-beta plus its propeptide) and is implicated in sequestering it in the extracellular matrix. Here we report the isolation of the mouse Ltbp-1 complementary DNA (cDNA) and gene. The longer form of the Ltbp-1 cDNA encodes a predicted 1713 amino acid protein containing 18 epidermal growth factor-like repeats, four 8-cysteine domains and several motifs that suggest interactions with alpha(IV)beta(1) and alpha(9)beta(1) integrins. Northern blotting analyses indicate that long and short Ltbp-1 transcripts are widely expressed in adult mouse tissues and most abundantly expressed in heart. Ltbp-1 is a single copy gene that maps to chromosome 17, band E (1-3) and encompasses more than 212 kb. The Ltbp-1 gene contains 34 exons and shows a similar organization to the LTBP-2 gene, suggesting that these genes originated from a common ancestral gene

Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at approximately 13 to 15 angstroms of the entry of tmRNA into the ribosome. The structure reveals how tmRNA could move through the ribosome despite its complicated topology and also suggests roles for proteins S1 and SmpB in the function of tmRNA

Human Flt3 ligand can expand dendritic cells (DC) and enhance immunogenicity in mice. However, little is known about the effects of murine Flt3 ligand (mFlt3L) on mouse DC development and function. We constructed a vector to transiently overexpress mFlt3L in mice. After a single treatment, up to 44% of splenocytes became CD11c(+) and the total number of DC increased 100-fold. DC expansion effects lasted for >35 days. mFlt3L DC were both phenotypically and functionally distinct. They had increased expression of MHC and costimulatory molecules and expressed elevated levels of B220 and DEC205 but had minimal CD4 staining. mFlt3L DC also had a markedly altered cytokine profile, including lowered secretion of IL-6, IL-10, IFN-gamma, and TNF-alpha, but had a slightly increased capacity to stimulate T cells in vitro. However, in a variety of in vivo models, DC expanded by mFlt3L induced tolerogenic effects on T cells. Adoptive transfer of Ag-pulsed mFlt3L splenic DC to naive mice actually caused faster rates of tumor growth and induced minimal CTL compared with control DC. mFlt3L also failed to protect against tumors in which human Flt3 ligand was protective, but depletion of CD4(+) T cells restored tumor protection. Our findings 1) demonstrate that mFlt3L has distinct effects on DC development, 2) suggest an important role for mFlt3L in generating DC that have tolerogenic effects on T cells, and 3) may have application in immunotherapy in generating massive numbers of DC for an extended duration

Dopaminergic (DA) neurons of substantia nigra in the midbrain control voluntary movement, and their degeneration is the cause of Parkinson's disease. The complete set of genes required to specifically determine the development of midbrain DA subgroups is not known yet. We report here that mice lacking the bicoid-related homeoprotein Pitx3 fail to develop DA neurons of the substantia nigra. Other mesencephalic DA neurons of the ventral tegmental area and retrorubral field are unaltered in their dopamine expression and histological organization. These data suggest that Pitx3-dependent gene expression is specifically required for the differentiation of DA progenitors within the mesencephalic DA system.

Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 Omega. cm(2). When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor-treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells were allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line that typically exhibits values below 800 omega x cm(2.) These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.

Recent studies have suggested glaucomatous loss of retinal ganglion cells and their axons in Alzheimer's disease. Amyloid beta peptides and phosphorylated tau protein have been implicated in the selective regional neuronal loss and protein accumulations characteristic of Alzheimer's disease. Similar protein accumulations are not present on glaucomatous retinal ganglion cells. Neurons die in both Alzheimer's disease and glaucoma by apoptosis, although the signaling pathways for neuronal degradation appear to differ in the two diseases. Alzheimer's disease features a loss of locus ceruleus noradrenergic neurons, which send axon terminals to the brain regions suffering neuronal apoptosis and results in reductions in noradrenaline in those regions. Activation of alpha-2 adrenergic receptors reduces neuronal apoptosis, in part through a protein kinase B (Akt)-dependent signaling pathway. Loss of noradrenaline innervation facilitates neuronal apoptosis in Alzheimer's disease models and may act similarly in glaucoma. Alpha-2 adrenergic receptor agonists offer the potential to slow the neuronal loss in both diseases by compensating for lost noradrenaline innervation

Beta-catenin regulates cell-cell adhesion and transduces signals from many pathways to regulate the transcriptional activities of Tcf/Lef DNA binding factors. Gene ablation and transgenic expression studies strongly support the concept that beta-catenin together with Lef/Tcf factors act as a switch to determine cell fate and promote cell survival and proliferation at several stages during mammary gland development. Mice expressing the negative regulator of Wnt/beta-catenin signaling (K14-Dkk) fail to form mammary buds, and those lacking Lef-1 show an early arrest in this process at stage E13.5. Stabilized deltaN89beta-catenin initiates precocious alveologenesis during pubertal development, and negative regulators of endogenous beta-catenin signaling suppress normal alveologenesis during pregnancy. Stabilized beta-catenin induces hyperplasia and mammary tumors in mice. Each of the beta-catenin-induced phenotypes is accompanied by upregulation of the target genes cyclin D1 and c-myc. Cyclin D1, however, is dispensable for tumor formation and the initiation of alveologenesis but is essential for later alveolar expansion

Putting together consistent cryo-EM structure, transient kinetic and mutant tRNA suppressor data, it appears that a deformed or waggling aminoacyl-tRNA is the critical transitional structure examined by the ribosome during decoding at the A site. The unusual conformation may be required for effective proofreading of the codon-anticodon complex

The 3beta-(2-diethylaminoethoxy)-androstenone HCl (U18666A), progesterone and several cationic amphiphilic drugs have been shown to alter the trafficking of a number of intracellular membrane proteins including CD63/Lamp-3, insulin growth factor 2/mannose 6-phosphate receptor (IGF2/MPR), and the Niemann-Pick C1 gene product (NPC1) as well as ganglioside GM1. We have examined the effects of these compounds on cultured melanocytes at concentrations that have been shown to effectively alter intracellular trafficking. Treatment of melanocytes with U18666A (2.5 micro M) or progesterone (15 micro M) for 96 h decreased melanin content an average of 67% as compared with control without lowering the total cellular tyrosinase activity. Steroidal alkaloids that preferentially act on the Sonic Hedgehog signaling pathway showed no related specificity in their ability to decrease pigmentation. In melanocytes treated with U18666A, tyrosinase accumulates in a compartment that contains both lysosome-associated membrane protein-1 (Lamp 1) and MPR, and stains with filipin, consistent with cholesterol-laden late endosomes/lysosomes. Our results suggest that tyrosinase, like the NPC1 gene product, traverses a U18666A-sensitive trafficking pathway