Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus.     

Electron microscopy studies on the quaternary structure of p53 reveal different binding modes for p53 tetramers in complex with DNA

The multidomain homotetrameric tumor suppressor p53 has two modes of binding dsDNA that are thought to be responsible for scanning and recognizing specific response elements (REs). The C termini bind nonspecifically to dsDNA. The four DNA-binding domains (DBDs) bind REs that have two symmetric 10 base-pair sequences. p53 bound to a 20-bp RE has the DBDs enveloping the DNA, which is in the center of the molecule surrounded by linker sequences to the tetramerization domain (Tet). We investigated by electron microscopy structures of p53 bound to DNA sequences consisting of a 20-bp RE with either 12 or 20 bp nonspecific extensions on either end. We found a variety of structures that give clues to recognition and scanning mechanisms. The 44- and 60-bp sequences gave rise to three and four classes of structures, respectively. One was similar to the known 20-bp structure, but the DBDs in the other classes were loosely arranged and incompatible with specific DNA recognition. Some of the complexes had density consistent with the C termini extending from Tet to the DNA, adjacent to the DBDs. Single-molecule fluorescence resonance energy transfer experiments detected the approach of the C termini towards the DBDs on addition of DNA. The structural data are consistent with p53 sliding along DNA via its C termini and the DNA-binding domains hopping on and off during searches for REs. The loose structures and posttranslational modifications account for the affinity of nonspecific DNA for p53 and point to a mechanism of enhancement of specificity by its binding to effector proteins.     

Role of T lymphocytes in angiotensin II-mediated microvascular thrombosis

Clinical trials and animal studies have revealed a role for the renin-angiotensin system in the enhanced thrombus development that is associated with hypertension. Because T lymphocytes have been implicated in the vascular dysfunction and blood pressure elevation associated with increased angiotensin II (Ang II) levels, we evaluated the role of the adaptive immune system in mediating the enhanced thrombosis during Ang II-induced hypertension. Light/dye-induced thrombosis was induced in cremaster arterioles of wild-type, immunodeficient Rag-1(-/-), CD8(+), or CD4(+) lymphocyte-deficient and NADPH oxidase (gp91(phox))-deficient mice implanted with an Ang II-loaded pump for 2 weeks. Chronic Ang II infusion enhanced arteriolar thrombosis in wild-type mice but not in Rag-1(-/-), CD4(+) T-cell-deficient, or gp91(phox-/-) mice. CD8(+) T-cell(-/-) mice exhibited partial protection. Adoptive transfer of T cells derived from wild-type or gp91(phox-/-) mice into Rag-1(-/-) restored the prothrombotic phenotype induced by Ang II. T lymphocytes (CD4(+) and, to a lesser extent, CD8(+)) play a major role in mediating the accelerated microvascular thrombosis associated with Ang II-induced hypertension. NADPH oxidase-derived reactive oxygen species, produced by cells other than T lymphocytes, also appear critical for the Ang II-enhanced, T cell-dependent thrombosis response.     

Microfabricated scaffold-guided endothelial morphogenesis in three-dimensional culture

Morphogenesis is a fundamental process by which new blood vessels are formed during angiogenesis. The ability to control angiogenesis would lead to improvements in tissue engineering constructions; indeed, the study of angiogenesis has numerous clinical applications, for example, in the investigation of metastatic cancer, peripheral and coronary vascular disease, and wound healing. Conventional in vitro organotypic cell culture approaches to these studies are limited primarily by their reliance on microvascular vessel formation through a random process of morphogenesis that lacks the spatial reproducibility and orientation needed for high-throughput drug testing. We have developed a bioreactor system for scaffold-guided tubulogenesis coupled with 3-D organotypic culture to spatially control vessel formation and its orientation. To create microchannels to guide microvessel formation, we fabricated rigid scaffolds using photolithography and light curing epoxy, and soft scaffolds formed by a polydimethylsiloxane (PDMS) stamp directly into collagen. Scaffolds seeded with dermal microvascular endothelial cells were placed between gelled layers of collagen containing dermal fibroblasts within a Transwell filter system and cultured for up to 2 weeks to allow for vessel maturation. Morphological analysis of thin tissue sections following standard histology and immunohistochemical detection of endothelial cells, fibroblasts, and basement membrane confirmed vessel formation along the microchannel walls with either scaffold. This system may also provide a means to explore revascularization within decellularized extracellular matrices, the culture of microvessel networks with controlled geometries, and possibly the spatial guidance of angiogenesis for interfacing with an external microfluidic supply network. As a new tool for guided angiogenesis, our approach introduces new possibilities for identification of anti-angiogenic therapeutics.     

Carfilzomib interacts synergistically with histone deacetylase inhibitors in mantle cell lymphoma cells in vitro and in vivo

Interactions between the proteasome inhibitor carfilzomib and the histone deacetylase (HDAC) inhibitors vorinostat and SNDX-275 were examined in mantle cell lymphoma (MCL) cells in vitro and in vivo. Coadministration of very low, marginally toxic carfilzomib concentrations (e.g., 3-4 nmol/L) with minimally lethal vorinostat or SNDX-275 concentrations induced sharp increases in mitochondrial injury and apoptosis in multiple MCL cell lines and primary MCL cells. Enhanced lethality was associated with c-jun-NH,-kinase (JNK) 1/2 activation, increased DNA damage (induction of λH2A.X), and ERK1/2 and AKT1/2 inactivation. Coadministration of carfilzomib and histone deacetylase inhibitors (HDACI) induced a marked increase in reactive oxygen species (ROS) generation and G(2)-M arrest. Significantly, the free radical scavenger tetrakis(4-benzoic acid) porphyrin (TBAP) blocked carfilzomib/HDACI-mediated ROS generation, λH2A.X formation, JNK1/2 activation, and lethality. Genetic (short hairpin RNA) knockdown of JNK1/2 significantly attenuated carfilzomib/HDACI-induced apoptosis, but did not prevent ROS generation or DNA damage. Carfilzomib/HDACI regimens were also active against bortezomib-resistant MCL cells. Finally, carfilzomib/vorinostat coadministration resulted in a pronounced reduction in tumor growth compared with single agent treatment in an MCL xenograft model associated with enhanced apoptosis, λH2A.X formation, and JNK activation. Collectively, these findings suggest that carfilzomib/HDACI regimens warrant attention in MCL.     

High throughput synthesis of extended pyrazolo[3,4-d]dihydropyrimidines

Thirteen 5-hetarylaminopyrazoles were synthesized in 62-93% yield through the arylation of 1-isopropyl- and 1-phenyl-5-aminopyrazoles with electrophilic hetarylhalides under optimized conditions. Condensation of 5-hetarylaminopyrazoles with carbonyl compounds facilitated by AcOH or Me(3)SiCl furnished 23 pyrazolo[3,4-d]dihydropyrimidines in 69-86% yield. The target compounds were isolated through simple crystallization. The scope and limitation of the method are discussed.