Faculty Bibliography

Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into several distinct lineages. Two key transcription factors, Runx2 and peroxisome proliferator-activated receptor gamma (PPARgamma), drive MSCs to differentiate into either osteoblasts or adipocytes, respectively. How these two transcription factors are regulated in order to specify these alternate cell fates remains a pivotal question. Here we report that a 14-3-3-binding protein, TAZ (transcriptional coactivator with PDZ-binding motif), coactivates Runx2-dependent gene transcription while repressing PPARgamma-dependent gene transcription. By modulating TAZ expression in model cell lines, mouse embryonic fibroblasts, and primary MSCs in culture and in zebrafish in vivo, we observed alterations in osteogenic versus adipogenic potential. These results indicate that TAZ functions as a molecular rheostat that modulates MSC differentiation.

Over 1 billion years ago, the animal kingdom diverged from the fungi. Nevertheless, a high sequence homology of 62% exists between human ribosomal protein S6 and S6A of Saccharomyces cerevisiae. To investigate whether this similarity in primary structure is mirrored in corresponding functional protein domains, the nuclear and nucleolar targeting signals were delineated in yeast S6A and compared to the known human S6 signals. The complete sequence of S6A and cDNA fragments was fused to the 5'-end of the LacZ gene, the constructs were transiently expressed in COS cells, and the subcellular localization of the fusion proteins was detected by indirect immunofluorescence. One bipartite and two monopartite nuclear localization signals as well as two nucleolar binding domains were identified in yeast S6A, which are located at homologous regions in human S6 protein. Remarkably, the number, nature, and position of these targeting signals have been conserved, albeit their amino acid sequences have presumably undergone a process of co-evolution with their corresponding rRNAs.

Self-assembly of a Gemini-shaped, chiral amphiphilic hexa-peri-hexabenzocoronene having two chiral oxyalkylene side chains, along with two lipophilic side chains, yields graphitic nanotubes with one-handed helical chirality. The nanotubes are characterized by an extremely high aspect ratio of >1,000 and have a uniform diameter of 20 nm and a wall thickness of 3 nm. The nanotubes with right- and left-handed helical senses were obtained from the (S)- and (R)-enantiomers of the amphiphile, respectively, due to an efficient translation of point chirality into supramolecular helical chirality. The (S)- and (R)-enantiomers coassemble at varying mole ratios to give nanotubes, whose circular dichroism profiles are almost unchanged over a wide range of the enantiomeric excess of the amphiphile (100-20%). The high level of chirality amplification thus observed indicates a long-range cooperativity in the self-assembling process. In sharp contrast, a hexabenzocoronene amphiphile with chiral lipophilic side chains did not form nanotubular assemblies. The present work demonstrates the majority rule in noncovalent systems and also may provide a synthetic strategy toward realization of molecular solenoids.

Phagocyte recognition and clearance of bacteria play essential roles in the host response to infection. In an on-going forward genetic screen, we identify the Drosophila melanogaster scavenger receptor Croquemort as a receptor for Staphylococcus aureus, implicating for the first time the CD36 family as phagocytic receptors for bacteria. In transfection assays, the mammalian Croquemort paralogue CD36 confers binding and internalization of Gram-positive and, to a lesser extent, Gram-negative bacteria. By mutational analysis, we show that internalization of S. aureus and its component lipoteichoic acid requires the COOH-terminal cytoplasmic portion of CD36, specifically Y463 and C464, which activates Toll-like receptor (TLR) 2/6 signaling. Macrophages lacking CD36 demonstrate reduced internalization of S. aureus and its component lipoteichoic acid, accompanied by a marked defect in tumor necrosis factor-alpha and IL-12 production. As a result, Cd36-/- mice fail to efficiently clear S. aureus in vivo resulting in profound bacteraemia. Thus, response to S. aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain, which initiates TLR2/6 signaling

BACKGROUND: We hypothesized that infertile men with varicoceles have molecular/genetic defects that interact with varicoceles to induce infertility. Studies directly on testis tissue appeared to be a way to link histology, markers for molecular/genetic defects and spermatogenesis, but testis biopsies may induce morbidity. In this report, we present safety and efficacy data on ultrasonically guided, single stick, percutaneous aspiration. METHODS: Biopsies were performed on 115 infertile men with varicoceles and five men with obstructive azoospermia. Morbidity was examined by pre- and post-biopsy ultrasound, efficacy by ability of two markers to predict >50% increase in sperm density post-operatively. All patients had three pre- and three post-operative semen analyses. RESULTS: 78.3% of patients had no ultrasonic testicular defects immediately post-biopsy. By 2 months, 100% had no defects. Biopsy markers [testicular cadmium (<0.453 ng/mg tissue) and an intact calcium channel mRNA sequence] predicted >50% increase in sperm density with 82.9 and 90.5% accuracy, respectively. CONCLUSIONS: Ultrasonically controlled, percutaneous aspiration testis biopsies are safe. Specimens so acquired can assist study of molecular/genetic markers associated with spermatogenesis in infertile men with varicoceles. Tissue cadmium level, calcium channel sequence and other markers may predict outcome of varicocele surgery

The role of hemodynamic forces and other signals from circulating blood in guiding the development of the retinal vasculature was examined by following the growth of these vessels in organ cultures. Retinal vascular development in organ cultures was monitored by immunofluorescent staining of retinal whole-mounts using antibodies against ICAM-2, a specific marker for endothelial cells and by vascular adenosine disphosphatase activity. Under culture conditions, the retinal vasculature from mice at postnatal day 3 (P3) grew from the optic nerve area to the edge of the retina in a manner similar to that observed in vivo. Both inner and outer vascular plexuses formed in retinal explants. Within the first few days of organ culture, the initial uniform meshwork of blood vessels was reorganized into arterioles, venules, and capillaries. As in animals, the initial retinal vascular plexus contained abundant vessels, and afterward some vessels regressed leading to the formation of a mature vascular bed. Changes in vascular density due to blood vessel growth and remodeling were confirmed by RT-PCR and Western blot analyses of ICAM-2 mRNA and protein levels, respectively. In addition, during in vitro retinal vascularization, arterioles acquired mural cell coverage, as shown by positive staining for alpha-smooth muscle actin. Thus, blood flow and blood-derived signals were not required for the development and maturation of retinal vessels. In contrast, stability of blood vessels in retinal explants was tightly regulated by endogenous levels of vascular endothelial growth factor-A (VEGF-A). VEGF-A was expressed in the explants throughout the culture period, and addition of neutralizing antibodies against VEGF-A to the organ culture caused a severe regression of blood vessels from the vascular front toward the optic nerve. In contrast, addition of anti-FGF-2 antibodies had no effect on the developing vasculature. Thus, retinal vascular development is dependent on local VEGF-A signals rather than systemic signals

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

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.