Faculty Bibliography

Comparing vertebrate genomes requires efficient cross-species sequence alignment programs. We describe COMBAT, a new mer-based method which can search rapidly for highly similar translated genomic sequences, with the stable-marriage algorithm with incomplete lists (SMI) as a filter scheme. We apply the COMBAT program to the comparative analysis of the human with the most recent bovine genome assemblies, and 84%similar to 95% of the homologous blocks identified by this program are confirmed by BLASTZ.

The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and skeletal muscle fibers. Essential for the formation of neuromuscular junction (NMJ) is the activation of MuSK, a muscle-specific receptor tyrosine kinase (DeChiara et al., 1996). In mice lacking MuSK, motor axons fail to stop and differentiate, acetylcholine receptors (AChRs) fail to cluster, and AChR genes are expressed uniformly in muscle (DeChiara et al., 1996; Gautam et al., 1996). The retrograde signals for presynaptic differentiation are not known. Because synapse-specific transcription, like presynaptic differentiation, is MuSK-dependent, it is possible that retrograde signals for presynaptic differentiation might be encoded by genes that are expressed preferentially by synaptic nuclei. To identify such synapse-specific genes we screened Affymetrix microarrays with RNA from the dissected, synapse-enriched, and extrasynaptic regions of skeletal muscle and further studied those genes that encode for the secreted or cell-surface proteins

The neurotrophins-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3 and NT-4-represent a family of proteins essential for neuronal survival and plasticity. Each neurotrophin can signal through two different transmembrane receptors, Trk receptor tyrosine kinases and the p75 neurotrophin receptor, the first member of the TNF receptor superfamily. Neurotrophic factors play an important role in neurodegenerative diseases, as well as neuropsychiatric disorders such as depression, bipolar disease and eating disorders. Indeed, a number of approaches have been taken to use neurotrophins to treat Alzheimer's dementia, amyotrophic lateral sclerosis and peripheral sensory neuropathy. However, many of these clinical trails have failed, due to problems in delivery and unforeseen side effects of neurotrophic factors. An alternative approach is to use ligands in the G protein-coupled receptor (GPCR) family to transactivate trophic activities. We have discovered that treatment with adenosine, a neuromodulator that acts through G protein-coupled receptors, is capable of activating Trk tyrosine kinase receptors. Transactivation of neurotrophic receptors by GPCR ligands raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of neurodegenerative diseases. This approach would allow for selective targeting of neurons that express specific G protein-coupled receptors and trophic factor receptors. GPCRs transduce information provided by extracellular signals to modulate synaptic activity and neurotransmission. In addition to the classical G protein signalling, GPCR ligands also activate receptor tyrosine kinases (RTK), including neurotrophin receptors. Activation of Trk neurotrophin receptors can occur by GPCR ligands in the absence of neurotrophins. Adenosine and PACAP (pituitary adenylate cyclase activating polypeptide) induce Trk activation specifically through their respective GPCRs to promote cell survival. Transactivation of Trks by GPCRs has emerged as a new theme in the biology of neurotrophin function. Although the precise role of transactivation is unknown, one possibility is that it adds a safety factor that might protect neurons from death in the absence of neurotrophins. Abnormal activity of the neurotrophin system has been implicated in several psychiatric and neurobiological illnesses. However, the lack of knowledge about the precise site of neurotrophin dysfunction has compromised the ability to improve the efficacy and the safety of drugs used in treatment modalities. If small-molecule GPCR ligands can ameliorate neuronal cell loss through Trk, transactivation may offer a new strategy for promoting trophic effects during neurodegeneration

Signal transduction down the Ras/MAPK pathway, including that critical to T cell activation, proliferation, and differentiation, has been generally considered to occur at the plasma membrane. It is now clear that the plasma membrane does not represent the only platform for Ras/MAPK signaling. Moreover, the plasma membrane itself is no longer considered a uniform structure but rather a patchwork of microdomains that can compartmentalize signaling. Signaling on internal membranes was first recognized on endosomes. Genetically encoded fluorescent probes for signaling events such as GTP/GDP exchange on Ras have revealed signaling on a variety of intracellular membranes, including the Golgi apparatus. In fibroblasts, Ras is activated on the plasma membrane and Golgi with distinct kinetics. The pathway by which Golgi-associated Ras becomes activated involves PLCgamma and RasGRP1 and may also require retrograde trafficking of Ras from the plasma membrane to the Golgi as a consequence of depalmitoylation. Thus, the Ras/MAPK pathway represents a clear example of compartmentalized signaling

Single-particle cryo-electron microscopy (cryo-EM) is the method of choice for studying the dynamics of macromolecular machines both at a phenomenological and, increasingly, at the molecular level, with the advent of high-resolution component X-ray structures and of progressively improving fitting algorithms. Cryo-EM has shed light on the structure of the ribosome during the four steps of translation: initiation, elongation, termination, and recycling. Interpretation of cryo-EM reconstructions of the ribosome in quasi-atomic detail reveals a picture in which the ribosome uses RNA not only to catalyze chemical reactions, but also as a means for signal transduction over large distances

Current treatments for damaged articular cartilage (i.e., shaving the articular surface, perforation or abrasion of the subchondral bone, and resurfacing with periosteal and perichondrial resurfacing) often produce fibrocartilage, or hyaline-appearing repair that is not sustained over time (Henche 1967, Ligament and Articular Cartilage Injuries. Springer-Verlag, New York, NY, pp. 157-164; Insall 1974, Clin. Orthop. 101: 61-67; Mitchell and Shepard 1976, J. Bone Joint Surg. [Am.] 58: 230-233; O'Driscoll et al. 1986, J. Bone Joint Surg. [Am.] 68: 1017-1035; 1989, Trans. Orthop. Res. Soc. 14: 145; Kim et al. 1991, J. Bone Joint Surg. [Am.] 73: 1301-1315). Autologous chondrocyte transplantation, although promising, requires two surgeries, has site-dependent and patient age limitations, and has unknown long-term donor site morbidity (Brittberg et al. 1994, N Engl. J. Med. 331: 889-895; Minas 2003, Orthopedics 26: 945-947; Peterson et al. 2003, J. Bone Joint Surg. Am. 85-A(Suppl. 2): S17-S24). Osteochondral allografts remain a widely used method of articular resurfacing to delay arthritic progression. The present study compared the histological response to four types of osteochondral implants in a rabbit model: autograft, frozen, freeze-dried, and fresh implants. Specimens implanted in the femoral groove were harvested at 6 and 12 weeks. Results showed similar restoration of the joint surface regardless of implant type, with a trend toward better repair at the later timepoint. As has been observed in other studies (Frenkel et al. 1997, J. Bone Joint Surg. 79B: 281-286; Toolan et al. 1998, J. Biomed. Mater. Res. 41: 244-250), each group in this study had at least one specimen in which a healthy-appearing surface on the implant was not well-integrated with host tissues. Although the differences were not statistically significant, freeze-dried implants at both timepoints had the best histological scores. The osteochondral grafts tested successfully restored the gross joint surface and congruity. At 12 weeks, no significant differences were observed between the various allografts and autologous osteochondral grafts

In recent years, investigators have made great progress in delineating developmental pathways of several lymphoid and myeloid lineages and in identifying transcription factors that establish and maintain their fate. However, the developmental branching points between these two large cell compartments are still controversial, and little is known about how their diversification is induced. Here, we give an overview of determinants that play a role at lymphoid-myeloid junctures, in particular transcription factors and cytokine receptors. Experiments showing that myeloid lineages can be reversibly reprogrammed into one another by transcription factor network perturbations are used to highlight key principles of lineage commitment. We also discuss experiments showing that lymphoid-to-myeloid but not myeloid-to-lymphoid conversions can be induced by the enforced expression of a single transcription factor. We close by proposing that this asymmetry is related to a higher complexity of transcription factor networks in lymphoid cells compared with myeloid cells, and we suggest that this feature must be considered when searching for mechanisms by which hematopoietic stem cells become committed to lymphoid lineages

Cells in the prospective somite of Xenopus laevis embryos rotate in an orchestrated manner to form a segregated somite. The prospective somite boundaries are prepatterned by gene expressions in the unsegmented presomitic mesoderm (PSM). However, the roles of polarized gene expression in this boundary formation are not well elucidated. Here we identified a novel gene, bowline, which localizes to the anterior halves of S-II, III in the PSM of X. laevis. Bowline associated with corepressor XGrg-4, a Xenopus homolog of Groucho/TLE protein. A WRPW tetrapeptide motif in Bowline was prerequisite for coprecipitation with XGrg-4 and for downregulation of X-Delta-2 by bowline RNA injection. This study indicates that Bowline is a novel protein interacting with Groucho/TLE and may play a role in somitogenesis in X. laevis.

Light intensity and atmospheric CO2 partial pressure are two environmental signals known to regulate stomatal numbers. It has previously been shown that if a mature Arabidopsis leaf is supplied with either elevated CO2 (750 ppm instead of ambient at 370 ppm) or reduced light levels (50 micromol m-2 s-1 instead of 250 micromol m-2 s-1), the young, developing leaves that are not receiving the treatment grow with a stomatal density as if they were exposed to the treatment. But the signal(s) that it is believed is generated in the mature leaves and transmitted to developing leaves are largely unknown. Photosynthetic rates of treated, mature Arabidopsis leaves increased in elevated CO2 and decreased when shaded, as would be expected. Similarly, the levels of sugars (glucose, fructose, and sucrose) in the treated mature leaves increased in elevated CO2 and decreased with shade treatment. The levels of sugar in developing leaves were also measured and it was found that they mirrored this result even though they were not receiving the shade or elevated CO2 treatment. To investigate the effect of these treatments on global gene expression patterns, transcriptomics analysis was carried out using Affymetrix, 22K, and ATH1 arrays. Total RNA was extracted from the developing leaves after the mature leaves had received either the ambient control treatment, the elevated CO2 treatment, or the shade treatment, or both elevated CO2 and shade treatments for 2, 4, 12, 24, 48, or 96 h. The experiment was replicated four times. Two other experiments were also conducted, one to compare and contrast gene expression in response to plants grown at elevated CO2 and the other to look at the effect of these treatments on the mature leaf. The data were analysed and 915 genes from the untreated, signalled leaves were identified as having expression levels affected by the shade treatment. These genes were then compared with those whose transcript abundance was affected by the shade treatment in the mature treated leaves (1181 genes) and with 220 putative 'stomatal signalling' genes previously identified from studies of the yoda mutant. The results of these experiments and how they relate to environmental signalling are discussed, as well as possible mechanisms for systemic signalling.