Faculty Bibliography

Nerve growth factor (NGF) functions as a ligand for two receptors, the TrkA tyrosine kinase receptor and the p75 neurotrophin receptor (p75NTR). The Ig-like domains of Trk receptors and the cysteine-rich repeats of p75NTR are involved in binding to the neurotrophins. Recently, a closely related gene to p75NTR called neurotrophin receptor homolog-2 (NRH2) was identified; however, the function of NRH2 and its relevance to neurotrophin signaling are unclear. NRH2 contains a similar transmembrane and intracellular domain as p75NTR but lacks the characteristic cysteine-rich repeats in the extracellular domain. Here we show that NRH2 is expressed in several neuronal populations that also express p75NTR and Trk receptors. NRH2 does not bind to NGF; however, coimmunoprecipitation experiments demonstrate that NRH2 is capable of interacting with TrkA receptors. Coexpression of NRH2 with TrkA receptors resulted in the formation of high-affinity binding sites for NGF. These results indicate that a transmembrane protein related to p75NTR is capable of modulating Trk receptor binding properties

We used a comparative genomic approach to identify putative cis-acting regulatory sequences of the zebrafish hoxb3a and hoxb4a genes. We aligned genomic sequences spanning the clustered Hoxb1 to Hoxb5 genes from pufferfish, mice, and humans with the zebrafish hoxba and hoxbb cluster sequences. We identified multiple blocks of conserved sequences in non-coding regions within and surrounding the Hoxb3/b4 gene locus; a subset of these blocks are conserved in the zebrafish hoxbb cluster, despite loss of hoxb3/b4 genes. Overall, we find that the architecture of the Hoxb3/b4 loci and of the conserved sequence elements is very similar in teleosts and mammals. Our analyses also revealed two alternative transcripts of the zebrafish hoxb3a gene and an exon sequence unusually located 10 kb upstream of adjacent hoxb4a; an equivalent murine Hoxb3 exon has not yet been confirmed. We show that many of the Hoxb3/b4 conserved non-coding sequences correlate with functional neural enhancers previously described in the mouse. Further, within the conserved non-coding sequences we have identified binding sites for transcription factors, including Kreisler/Valentino, Krox20, Hox, and Pbx, some of which had not been previously described for the mouse. Finally, we demonstrate that the regulatory sequences of zebrafish hoxa3a are divergent with respect to the mouse ortholog Hoxa3, or the paralog hoxb3a. Despite limited conservation of regulatory sequences, zebrafish hoxa3a and hoxb3a genes share very similar expression profiles

AC133 is a member of a novel family of cell surface proteins with 5 transmembrane domains. The function of AC133 is unknown. Although AC133 mRNA is detected in different tissues, its expression in the hematopoietic system is restricted to CD34+ stem cells. AC133 is also expressed on stem cells of other tissues, including endothelial progenitor cells. However, despite the potential importance of AC133 to the field of stem cell biology, nothing is known about the transcriptional regulation of AC133 expression. In this report we showed that the human AC133 gene has at least 9 distinctive 5'-untranslated region (UTR) exons, resulting in the formation of at least 7 alternatively spliced 5'-UTR isoforms of AC133 mRNA, which are expressed in a tissue-dependent manner. We found that transcription of these AC133 isoforms is controlled by 5 alternative promoters, and we demonstrated their activity on AC133-expressing cell lines using a luciferase reporter system. We also showed that in vitro methylation of 2 of these AC133 promoters completely suppresses their activity, suggesting that methylation plays a role in their regulation. Identification of tissue-specific AC133 promoters may provide a novel method to isolate tissue-specific stem and progenitor cells

At central synapses, P/Q-type Ca(2+) channels normally provide a critical Ca(2+) entry pathway for neurotransmission. Nevertheless, we found that nerve terminals lacking alpha(1A) (Ca(V)2.1), the pore-forming subunit of P/Q-type channels, displayed a remarkable preservation of synaptic function. Two consistent physiological changes reflective of synaptic homeostasis were observed in cultured hippocampal neurons derived from alpha(1A) (-/-) mice. First, the presynaptic response to an ionophore-mediated Ca(2+) elevation was 50% greater, indicating an enhanced Ca(2+) sensitivity of the release machinery. Second, basal miniature excitatory postsynaptic current frequency in alpha(1A) (-/-) neurons was increased 2-fold compared with WT neurons and occluded the normal response of presynaptic terminals to cAMP elevation, suggesting that the compensatory mechanism in alpha(1A) (-/-) synapses and the modulation of presynaptic function by PKA might share a final common pathway. We used cDNA microarray analysis to identify molecular changes underlying homeostatic regulation in the alpha(1A) (-/-) hippocampus. The 40,000 entries in our custom-made array included likely targets of presynaptic homeostasis, along with many other transcripts, allowing a wide-ranging examination of gene expression. The developmental pattern of changes in transcript levels relative to WT was striking; mRNAs at 5 and 11 days postnatal showed little deviation, but clear differences emerged by 22 days. Many of the transcripts that differed significantly in abundance corresponded to known genes that could be incorporated within a logical pattern consistent with the modulation of presynaptic function. Changes in endocytotic proteins, signal transduction kinases, and candidates for Ca(2+)-sensing molecules were consistent with implications of the direct physiological experiments

In both animals and plants, many developmentally important regulatory genes have complementary microRNAs (miRNAs), which suggests that these miRNAs constitute a class of developmental signalling molecules. Leaves of higher plants exhibit a varying degree of asymmetry along the adaxial/abaxial (upper/lower) axis. This asymmetry is specified through the polarized expression of class III homeodomain/leucine zipper (HD-ZIPIII) genes. In Arabidopsis, three such genes, PHABULOSA (PHB), PHAVOLUTA (PHV) and REVOLUTA (REV), are expressed throughout the incipient leaf, but become adaxially localized after primordium emergence. Downregulation of the HD-ZIPIII genes allows expression of the KANADI and YABBY genes, which specify abaxial fate. PHB, PHV and REV transcripts contain a complementary site for miRNA165 and miRNA166, which can direct their cleavage in vitro. Here we show that miRNA166 constitutes a highly conserved polarizing signal whose expression pattern spatially defines the expression domain of the maize hd-zipIII family member rolled leaf1 (rld1). Moreover, the progressively expanding expression pattern of miRNA166 during leaf development and its accumulation in phloem suggests that miRNA166 may form a movable signal that emanates from a signalling centre below the incipient leaf.

This paper explores the use of particle filters, rooted in Bayesian estimation, as a device for tracking statistical variations in the channel matrix of a narrowband multiple-input, multiple-output (MIMO) wireless channel. The motivation is to permit the receiver acquire channel state information through a semiblind strategy and thereby improve the receiver performance of the wireless communication system. To that end, the paper compares the particle filter as well as an improved version of the particle filter using gradient information, to the conventional Kalman filter and mixture Kalman filter with two metrics in mind: receiver performance curves and computational complexity. The comparisons, also including differential phase modulation, are carried out using real-life recorded MIMO wireless data.

Osteoblasts exhibit a more differentiated morphology on surfaces with rough microtopographies. Surface effects are often mediated through integrins that bind the RGD motif in cell attachment proteins. Here, we tested the hypothesis that modulating access to RGD binding sites can modify the response of osteoblasts to surface microtopography. MG63 immature osteoblast-like cells were cultured on smooth (Ti sputter-coated Si wafers) and rough (grit blasted/acid etched) Ti surfaces that were modified with adsorbed monomolecular layers of a comb-like graft copolymer, poly-(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), to limit nonspecific protein adsorption. PLL-g-PEG coatings were functionalized with varying amounts of an integrin-receptor-binding RGD peptide GCRGYGRGDSPG (PLL-g-PEG/PEG-RGD) or a nonbinding RDG control sequence GCRGYGRDGSPG (PLL-g-PEG/PEG-RDG). Response to PLL-g-PEG alone was compared with response to surfaces on which 2-18% of the polymer sidechains were functionalized with the RGD peptide or the RDG peptide. To examine RGD dose-response, peptide surface concentration was varied between 0 and 6.4 pmol/cm(2). In addition, cells were cultured on uncoated Ti or Ti coated with PLL-g-PEG or PLL-g-PEG/PEG-RGD at an RGD surface concentration of 0.7 pmol/cm(2), and free RGDS was added to the media to block integrin binding. Analyses were performed 24 h after cultures had achieved confluence on the tissue culture plastic surface. Cell number was reduced on smooth Ti compared to plastic or glass and further decreased on surfaces coated with PLL-g-PEG or PLL-g-PEG/PEG-RDG, but was restored to control levels when PLL-g-PEG/PEG-RGD was present. Alkaline phosphatase specific activity and osteocalcin levels were increased on PLL-g-PEG alone or PLL-g-PEG/PEG-RDG, but PLL-g-PEG/PEG-RGD reduced the parameters to control levels. On rough Ti surfaces, cell number was reduced to a greater extent than on smooth Ti. PLL-g-PEG coatings reduced alkaline phosphatase and increased osteocalcin in a manner that was synergistic with surface roughness. The RDG peptide did not alter the PLL-g-PEG effect but the RGD peptide restored these markers to their control levels. PLL-g-PEG coatings also increased TGF-beta1 and PGE(2) in conditioned media of cells cultured on smooth or rough Ti; there was a 20x increase on rough Ti coated with PLL-g-PEG. PLL-g-PEG effects were inhibited dose dependently by addition of the RGD peptide to the surface. Free RGDS did not decrease the effect elicited by PLL-g-PEG surfaces. These unexpected results suggest that PLL-g-PEG may have osteogenic properties, perhaps correlated with effects that alter cell attachment and spreading, and promote a more differentiated morphology.

Gene expression in skeletal muscle is regulated by a family of myogenic basic helix-loop-helix (bHLH) proteins. The binding of these bHLH proteins, notably MyoD and myogenin, to E-boxes in their own regulatory regions is blocked by protein kinase C (PKC)-mediated phosphorylation of a single threonine residue in their basic region. Because electrical stimulation increases PKC activity in skeletal muscle, these data have led to an attractive model suggesting that electrical activity suppresses gene expression by stimulating phosphorylation of this critical threonine residue in myogenic bHLH proteins. We show that electrical activity stimulates phosphorylation of myogenin at threonine 87 (T87) in vivo and that calmodulin-dependent kinase II (CaMKII), as well as PKC, catalyzes this reaction in vitro. We find that phosphorylation of myogenin at T87 is dispensable for skeletal muscle development. We show, however, that the decrease in myogenin (myg) expression following innervation is delayed and that the increase in expression following denervation is accelerated in mutant mice lacking phosphorylation of myogenin at T87. These data indicate that two distinct innervation-dependent mechanisms restrain myogenin activity: an inactivation mechanism mediated by phosphorylation of myogenin at T87, and a second, novel regulatory mechanism that regulates myg gene activity independently of T87 phosphorylation