Faculty Bibliography

We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L. A. Chasin, Genes Dev. 18:1241-1250, 2004). When inserted into a poorly spliced test exon, all eight tested octamers stimulated splicing, a result consistent with their identification as exonic splicing enhancers (ESEs). Here we present a much more stringent test of the validity of this list of PESEs. Twenty-two naturally occurring examples of nonoverlapping PESEs or PESE clusters were identified in six mammalian exons; five of the six exons tested are constitutively spliced. Each of the 22 individual PESEs or PESE clusters was disrupted by site-directed mutagenesis, usually by a single-base substitution. Eighteen of the 22 disruptions (82%) resulted in decreased splicing efficiency. In contrast, 24 control mutations had little or no effect on splicing. This high rate of success suggests that most PESEs function as ESEs in their natural context. Like most exons, these exons contain several PESEs. Since knocking out any one of several could produce a severalfold decrease in splicing efficiency, we conclude that there is little redundancy among ESEs in an exon and that they must work in concert to optimize splicing.

Three tandemly arrayed protocadherin gene clusters (Pcdh-alpha, -beta, -gamma) comprising more than 50 genes are found in human and mouse. Here, we have investigated the expression and distribution of individual gamma-protocadherins (Pcdhs-gamma) in the developing mouse brain. We find that transfection of Pcdh-gamma genes promotes calcium-dependent cell adhesion in HEK 293 cells. Furthermore, Pcdh-gamma can be recruited to synapses of transfected primary hippocampal neurons. Several individual members of the in total 22 Pcdhs-gamma were chosen to examine the expression of the three subfamilies, Pcdh-gammaA, -gammaB, and -gammaC. These Pcdh-gamma transcripts are expressed all over the brain, with minor regional and cell-type specific differences. Interestingly, a distinct, later onset of expression is observed for Pcdh-gammaC5, a gene located at the end of the Pcdh-gamma cluster. Largely overlapping expression patterns of individual Pcdh-gamma proteins are detected with anti-peptide antibodies. Small differences are observed in the staining of dendritic processes and synapse-rich layers. Our results support the idea that Pcdhs-gamma participate in neuronal differentiation and may be implicated in the fine-tuning of neuronal morphology and synaptogenesis. Cell autonomous regulation of transcription might generate the widespread distribution of individual Pcdhs-gamma in the brain, which is strikingly different from the restricted expression patterns observed for classical cadherins. Thus, a defined set of Pcdhs-gamma may engage in neuronal adhesion and signaling on the cellular level.

Latent transforming growth factor (TGF)-beta binding proteins (LTBPs) modulate the secretion and activation of latent TGF-beta. To explore LTBP function in vivo, we created an Ltbp-3(-/-) mouse that has developmental emphysema with decreased septation in terminal alveoli. Differences in distal airspace enlargement were obvious at day 6 after birth. Secondary septation was inhibited, so by days 21 to 28 the mean linear intercept was approximately twofold greater in mutant versus control lungs. There were no differences in lung collagen and elastin, visualized by immunohistochemistry, or in myofibroblast numbers, determined by alpha-smooth muscle actin-positive cells, between mutant or wild-type lungs as the animals aged, other than differences associated with altered lung structure in mutant animals. However, from day 10 there was twice the number of alveolar type II cells in mutant alveoli compared to controls. At days 6 and 10, a transient enhancement in cell proliferation in the mutant lungs was observed by both 5-bromo-2'-deoxy-uridine and proliferating cell nuclear antigen labeling, accompanied by enhanced numbers of terminal dUTP nick-end labeling-positive cells at days 4, 6, and 10. Finally, there was a transient decrease in TGF-beta signaling at days 4 to 6 in Ltbp-3(-/-) lungs. These results indicate that in the absence of Ltbp-3, a temporary decrease in TGF-beta signaling in the lungs at days 4 to 6 alters cell proliferation, correlating with inhibition of septation and developmental emphysema

Extensor mechanism disruption from quadriceps tendon rupture, patellar fracture, or patellar tendon rupture is an uncommon complication of total knee arthroplasty. Extensor mechanism disruption can occur either intraoperatively or postoperatively. Common intraoperative causes include avulsion or tendon injury arising from excessive tension during surgical exposure, improper patellar resection, and devascularization due to injudicious lateral retinacular release or multiple prior surgeries. The usual postoperative causes are tissue necrosis arising from infection, component malalignment, and trauma. A wide range of treatment options is available for managing these difficult problems, and recent advances in alternative techniques for reconstruction have yielded promising results

Ras proteins associate with cellular membranes by virtue of a series of post-translational modifications of their C-terminal CAAX sequences. The discovery that two of the three enzymes that modify CAAX proteins are restricted to the endoplasmic reticulum led to the recognition that all nascent Ras proteins transit endomembranes en route to the PM (plasma membrane) and that at steady-state N-Ras and H-Ras are highly expressed on the Golgi apparatus. To test the hypothesis that Ras proteins on internal membranes can signal, we developed a fluorescent probe that reports when and where in living cells Ras becomes active. We found that growth factors stimulated rapid and transient activation of Ras on the PM followed by delayed and sustained activation on the Golgi. We mapped one pathway responsible for this activity as involving PLCgamma (phospholipase Cgamma)/DAG (diacylglycerol)+Ca2+/RasGRP1. Using mammalian cells and fission yeast, we have shown that differential localization of activated Ras preferentially activates distinct signalling pathways. In very recent work, we have found that (i) the subcellular localization of K-Ras can be acutely modulated by phosphorylation of its C-terminal hypervariable region by PKC, (ii) among the membranes upon which phosphorylated K-Ras accumulates is the outer mitochondrial membrane and (iii) phosphorylated, internalized K-Ras promotes apoptosis. Thus the signalling output of Ras depends on its subcellular localization

For sperm to fertilize eggs, they must first bind to the thick zona pellucida (ZP) that surrounds the plasma membrane of all unfertilized mammalian eggs. An extensive literature suggests that mouse sperm recognize and bind to a specific ZP glycoprotein called mZP3. However, the role of individual ZP glycoproteins in binding of mouse sperm to eggs has been called into question by recent transgenic experiments with null mice. Results of such experiments have been interpreted to mean that binding of sperm depends on the supramolecular structure of the ZP, not on an individual ZP glycoprotein. Here, it is argued that results of these transgenic experiments actually are consistent with the prevailing view of gamete recognition that implicates a specific ZP glycoprotein in both binding of mouse sperm to eggs and induction of the acrosome reaction.

Several major diseases of old age, including atherosclerosis, macular degeneration and neurodegenerative diseases are associated with the intracellular accumulation of substances that impair cellular function and viability. Moreover, the accumulation of lipofuscin, a substance that may have similarly deleterious effects, is one of the most universal markers of aging in postmitotic cells. Reversing this accumulation may thus be valuable, but has proven challenging, doubtless because substances resistant to cellular catabolism are inherently hard to degrade. We suggest a radically new approach: augmenting humans' natural catabolic machinery with microbial enzymes. Many recalcitrant organic molecules are naturally degraded in the soil. Since the soil in certain environments - graveyards, for example - is enriched in human remains but does not accumulate these substances, it presumably harbours microbes that degrade them. The enzymes responsible could be identified and engineered to metabolise these substances in vivo. Here, we survey a range of such substances, their putative roles in age-related diseases and the possible benefits of their removal. We discuss how microbes capable of degrading them can be isolated, characterised and their relevant enzymes engineered for this purpose and ways to avoid potential side-effects.

The prevention of new blood vessel growth is an increasingly attractive strategy to limit tumor growth. However, it remains unclear whether anti-angiogenesis approaches will impair wound healing, a process thought to be angiogenesis dependent. Results of previous studies differ as to whether angiogenesis inhibitors delay wound healing. We evaluated whether endostatin at tumor-inhibiting doses delayed excisional wound closure. C57/BL6J mice were treated with endostatin or phosphate-buffered solution 3 days prior to the creation of two full-thickness wounds on the dorsum. Endostatin was administered daily until wound closure was complete. A third group received endostatin, but also had daily topical vascular endothelial growth factor applied locally to the wound. Wound area was measured daily and the wounds were analyzed for granulation tissue formation, epithelial gap, and wound vascularity. Endostatin-treated mice showed a significant delay in wound healing. Granulation tissue formation and wound vascularity were significantly decreased, but reepithelialization was not effected. Topical vascular endothelial growth factor application to wounds in endostatin-treated mice resulted in increased granulation tissue formation, increased wound vascularity, and wound closure approaching that of control mice. This study shows that the angiogenesis inhibitor endostatin delays wound healing and that topical vascular endothelial growth factor is effective in counteracting this effect

Macrophage internalization of modified lipoproteins is thought to play a critical role in the initiation of atherogenesis. Two scavenger receptors, scavenger receptor A (SR-A) and CD36, have been centrally implicated in this lipid uptake process. Previous studies showed that these receptors mediated the majority of cholesterol ester accumulation in macrophages exposed to oxidized LDL and that mice with deletions of either receptor exhibited marked reductions in atherosclerosis. This work has contributed to an atherosclerosis paradigm: scavenger receptor-mediated oxidized lipoprotein uptake is required for foam cell formation and atherogenesis. In this study, Apoe-/- mice lacking SR-A or CD36, backcrossed into the C57BL/6 strain for 7 generations, were fed an atherogenic diet for 8 weeks. Hyperlipidemic Cd36-/-Apoe-/- and Msr1-/-Apoe-/- mice showed significant reductions in peritoneal macrophage lipid accumulation in vivo; however, in contrast with previous reports, this was associated with increased aortic sinus lesion areas. Characterization of aortic sinus lesions by electron microscopy and immunohistochemistry showed abundant macrophage foam cells, indicating that lipid uptake by intimal macrophages occurs in the absence of CD36 or SR-A. These data show that alternative lipid uptake mechanisms may contribute to macrophage cholesterol ester accumulation in vivo and suggest that the roles of SR-A and CD36 as proatherosclerotic mediators of modified LDL uptake in vivo need to be reassessed