Faculty Bibliography

Irradiation of an amorphous layer of dried sodium phosphate buffer (pH = 7.0) by transmission electron microscopy (100-120 kV) causes rapid formation of numerous small spherical bubbles [10-100 A (= 1-10 nm)] containing an unknown gas. Bubbling is detected even with the first low-dose exposure. In a thin layer (ca. 100-150 A), bubbling typically goes through nucleation, growth, possible fusion, and end-state, after which further changes are not apparent; co-irradiated adjacent areas having a slightly smaller thickness never develop bubbles. In moderately thicker regions (ca. over 200 A), there is no end-state. Instead, a complex sequence of microstructural changes is elicited during continued intermittent high-dose irradiation: nucleation, growth, early simple fusions, a second round of extensive multiple fusions, general reduction of matrix thickness (producing flattening and expansion of larger bubbles, occasional bubble fission, and formation of very large irregularly-shaped bubbles by a third round of compound fusion events), and slow shrinkage of all bubbles. The ongoing lighter appearance of bubble lumens, maintenance of their rounded shape, and extensive changes in size and form indicate that gas content continues throughout their surprisingly long lifetime; the thin dense boundary layer surrounding all bubbles is proposed to be the main mechanism for their long lifetime.

The field of tissue engineering has made considerable strides since it was first described in the late 1980s. The advent and subsequent boom in stem cell biology, emergence of novel technologies for biomaterial development and further understanding of developmental biology have contributed to this accelerated progress. However, continued efforts to translate tissue-engineering strategies into clinical therapies have been hampered by the problems associated with scaling up laboratory methods to produce large, complex tissues. The significant challenges faced by tissue engineers include the production of an intact vasculature within a tissue-engineered construct and recapitulation of the size and complexity of a whole organ. Here we review the basic components necessary for bioengineering organs-biomaterials, cells and bioactive molecules-and discuss various approaches for augmenting these principles to achieve organ level tissue engineering. Ultimately, the successful translation of tissue-engineered constructs into everyday clinical practice will depend upon the ability of the tissue engineer to "scale up" every aspect of the research and development process.

The glucuronide transporter GusB, the product of the gusB gene from Escherichia coli, is responsible for detoxification of metabolites. In this study, we successfully expressed GusB homologously in E. coli and investigated its oligomeric state in n-dodecyl-beta-D: -maltoside (DDM) detergent solution. Evidence for a pentameric state with a Stokes radius of 57 +/- 2 A for the purified GusB protein in DDM solution was obtained by analytical size-exclusion HPLC. The elution peak corresponding to pentameric GusB is commonly seen in elution profiles in the different buffer systems examined over a wide pH range. Hence, it is likely that GusB resides in the membrane as a pentamer. Stability studies with different incubation periods with the typical lipids, such as dimyristoylphosphatidylcholine, and total E. coli phospholipids, as the representatives of both phosphatidylcholine and phosphatidylethanolamine, show some clues to two-dimensional crystallization of GusB with lipids.

Activation of the c-JUN N-terminal kinase (JNK) pathway is implicated in a number of important physiological processes, from embryonic morphogenesis to cell survival and apoptosis. JNK stimulatory phosphatase 1 (JSP1) is a member of the dual-specificity phosphatase subfamily of protein tyrosine phosphatases. In contrast to other dual-specificity phosphatases that catalyze the inactivation of mitogen-activated protein kinases, expression of JSP1 activates JNK-mediated signaling. JSP1 and its relative DUSP15 are unique among members of the protein tyrosine phosphatase family in that they contain a potential myristoylation site at the N-terminus (MGNGMXK). In this study, we investigated whether JSP1 was myristoylated and examined the functional consequences of myristoylation. Using mass spectrometry, we showed that wild-type JSP1, but not a JSP1 mutant in which Gly2 was mutated to Ala (JSP1-G2A), was myristoylated in cells. Although JSP1 maintained intrinsic phosphatase activity in the absence of myristoylation, the subcellular localization of the enzyme was altered. Compared with the wild type, the ability of nonmyristoylated JSP1 to induce JNK activation and phosphorylation of the transcription factor c-JUN was attenuated. Upon expression of wild-type JSP1, a subpopulation of cells, with the highest levels of the phosphatase, was induced to float off the dish and undergo apoptosis. In contrast, cells expressing similar levels of JSP1-G2A remained attached, further highlighting that the myristoylation mutant was functionally compromised.

Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 +/- 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% +/- 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2 degrees structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in "Candidatus Protochlamydia amoebophila." Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.

The mammary gland is an epidermal appendage that begins to form during embryogenesis, but whose development is only completed during pregnancy. Each mammary gland begins as a budlike invagination of the surface ectoderm, which then gives rise to a simple duct system by birth. Subsequent development occurs during sexual maturation and during pregnancy and lactation. In this review, we outline the distinct stages of embryonic mammary development and discuss the molecular pathways involved in the regulation of morphogenesis at each stage. We also discuss the potential relevance of embryonic breast development to the pathophysiology of breast cancer and highlight questions for future research

PURPOSE: To evaluate the recovery of the gastrointestinal tract in lethally irradiated mice treated with human cord blood and antibiotics. MATERIALS AND METHODS: A/J mice were randomly assigned to seven study groups, including groups exposed to acute 9 Gy from (137)Cs gamma-rays to the whole body. Four hours after irradiation, exposed mice were treated with either cord blood nucleated cells, Levaquin, or a combination of both. Weight gain/loss and survival were monitored for 2 months. Upon death or euthanasia, the organs were prepared for molecular and histological analyses. RESULTS: Whereas irradiated mice (n = 9) lived 6-15 days, approximately 60% of irradiated mice that received the combined treatment (n = 7) survived more than 50 days. None of the treated animals developed Graft versus Host disease. All animals lost weight after irradiation; however, the 50(+) days-survivors (n = 4) gained on average approximately 1.8 g over their initial weight. Whereas hemorrhagic bone marrow and large areas of transmural necrosis were observed in the bowel of the irradiated mice, the 50(+) days-survivors showed recovery of the bone marrow. They behaved normally and had significant but incomplete recovery of the intestinal and colonic mucosa. Human DNA was detected in their organs, particularly in the large intestine. CONCLUSION: Red cell-depleted cord blood transfusions combined with antibiotic treatment contribute to bone marrow and gastrointestinal recovery in high dose-irradiated mice, and may be an available therapy for mass casualties during radiological emergencies.

Bone morphogenetic proteins (BMPs) participate in multiple stages of the fetal skeletogenic program from promoting cell condensation to regulating chondrogenesis and bone formation through endochondral ossification. Here, we show that these pleiotropic functions are recapitulated when recombinant BMPs are used to augment skeletal tissue repair. In addition to their well-documented ability to stimulate chondrogenesis in a skeletal injury, we show that recombinant BMPs (rBMPs) simultaneously suppress the differentiation of skeletal progenitor cells in the endosteum and bone marrow cavity to an osteoblast lineage. Both the prochondrogenic and antiosteogenic effects are achieved because rBMP inhibits endogenous beta-catenin-dependent Wnt signaling. In the injured periosteum, this repression of Wnt activity results in sox9 upregulation; consequently, cells in the injured periosteum adopt a chondrogenic fate. In the injured endosteum, rBMP also inhibits Wnt signaling, which results in the runx2 and collagen type I downregulation; consequently, cells in this region fail to differentiate into osteoblasts. In muscle surrounding the skeletal injury site, rBMP treatment induces Smad phosphorylation followed by exuberant cell proliferation, an increase in alkaline phosphatase activity, and chondrogenic differentiation. Thus different populations of adult skeletal progenitor cells interpret the same rBMP stimulus in unique ways, and these responses mirror the pleiotropic effects of BMPs during fetal skeletogenesis. These mechanistic insights may be particularly useful for optimizing the reparative potential of rBMPs while simultaneously minimizing their adverse outcomes.

High density lipoprotein (HDL), an endogenous nanoparticle, transports fat throughout the body and is capable of transferring cholesterol from atheroma in the vessel wall to the liver. In the present study, we utilized HDL as a multimodal nanoparticle platform for tumor targeting and imaging via nonspecific accumulation and specific binding to angiogenically activated blood vessels. We reconstituted HDL (rHDL) with amphiphilic gadolinium chelates and fluorescent dyes. To target angiogenic endothelial cells, rHDL was functionalized with alphavbeta3-integrin-specific RGD peptides (rHDL-RGD). Nonspecific RAD peptides were conjugated to rHDL nanoparticles as a control (rHDL-RAD). It was observed in vitro that all 3 nanoparticles were phagocytosed by macrophages, while alphavbeta3-integrin-specific rHDL-RGD nanoparticles were preferentially taken up by endothelial cells. The uptake of nanoparticles in mouse tumors was evaluated in vivo using near infrared (NIR) and MR imaging. All nanoparticles accumulated in tumors but with very different accumulation/binding kinetics as observed by NIR imaging. Moreover, confocal microscopy revealed rHDL-RGD to be associated with tumor endothelial cells, while rHDL and rHDL-RAD nanoparticles were mainly found in the interstitial space. This study demonstrates the ability to reroute HDL from its natural targets to tumor blood vessels and its potential for multimodal imaging of tumor-associated processes.

In Drosophila, germ cell survival and directionality of migration are controlled by two lipid phosphate phosphatases (LPP), wunen (wun) and wunen-2 (wun2). wun wun2 double mutant analysis reveals that the two genes, hereafter collectively called wunens, act redundantly in primordial germ cells. We find that wunens mediate germ cell-germ cell repulsion and that this repulsion is necessary for germ cell dispersal and proper transepithelial migration at the onset of migration and for the equal sorting of the germ cells between the two embryonic gonads during their migration. We propose that this dispersal function optimizes adult fecundity by assuring maximal germ cell occupancy of both gonads. Furthermore, we find that the requirement for wunens in germ cell survival can be eliminated by blocking germ cell migration. We suggest that this essential function of Wunen is needed to maintain cell integrity in actively migrating germ cells