Acknowledgments

One of the major causes of worldwide amphibian declines is a skin infection caused by a pathogenic chytrid fungus (Batrachochytrium dendrobatidis). This study documents the interactions between this pathogen and a susceptible amphibian host, the boreal toad (Bufo boreas). The amount of time following exposure until death is influenced by the dosage of infectious zoospores, duration of exposure, and body size of the toad. The significant relation between dosage and the number of days survived (dose-response curve) supports the hypothesis that the degree of infection must reach a particular threshold of about 10⁷–10⁸ zoosporangia before death results. Variation in air temperature between 12°C and 23°C had no significant effect on survival time. The infection can be transmitted from infected to healthy animals by contact with water containing zoospores; no physical contact between animals is required. These results are correlated with observations on the population biology of boreal toads in which mortalities associated with B. dendrobatidis have been identified.     

Bone marrow-derived endothelial progenitor cells contribute to the angiogenic switch in tumor growth and metastatic progression

Emerging evidence indicates that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated growth of certain tumors in mice and human. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. While the contributions of EPCs to neovessel formation in spontaneous and transplanted tumors and to the metastatic transition have been reported to be relatively low, remarkably, specific EPC ablation in vivo has resulted in severe angiogenesis inhibition and impaired primary and metastatic tumor growth. The existence of a BM reservoir of EPCs, and the selective involvement of EPCs in neovascularization, have attracted considerable interest because these cells represent novel target for therapeutic intervention. In addition, EPCs are also being used as pharmacodynamic surrogate markers for monitoring cancer progression, as well as for optimizing efficacy of anti-angiogenic therapies in the clinic. This review will focus primarily on recent advances and emerging concepts in the field of EPC biology and discuss ongoing debates involving the role of EPCs in tumor neovascularization. For detailed information on the in vitro characterization of EPCs contribution to non-tumor pathologies, the reader is directed towards several excellent reviews and publications [F. Bertolini, Y. Shaked, P. Mancuso and R.S. Kerbel, Nat. Rev., Cancer 6 (2006) 835–845. [1]] [J.M. Hill, T. Finkel and A.A. Quyyumi, Vox Sang. 87 Suppl 2 (2004) 31–37. [2]] [A.Y. Khakoo and T. Finkel, Annu. Rev. Med. 56 (2005) 79–101. [3]] [H.G. Kopp, C.A. Ramos and S. Rafii, Curr. Opin. Hematol. 13 (2006) 175–181. [4]; K.K. Hirschi, D.A. Ingram and M.C. Yoder, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 1584–1595. [5]; F. Timmermans, J. Plum, M.C. Yoder, D.A. Ingram, B. Vandekerckhove and J. Case, J. Cell. Mol. Med. 13 (2009) 87–102. [6]] and reviews by Bertolini, Voest and Yoder in this issue.     

Metal ion and DNA binding by single-chain <Emphasis Type="Italic">Pvu</Emphasis>II endonuclease: lessons from the linker

Understanding the roles of metal ions in restriction enzymes has been complicated by both the presence of two metal ions in many active sites and their homodimeric structure. Using a single-chain form of the wild-type restriction enzyme PvuII (scWT) in which subunits are fused with a short polypeptide linker (Simoncsits et al. in J. Mol. Biol. 309:89–97, 2001), we have characterized metal ion and DNA binding behavior in one subunit and examined the effects of the linker on dimer behavior. scWT exhibits heteronuclear single quantum coherence NMR spectra similar to those of native wild-type PvuII (WT). For scWT, isothermal titration calorimetry data fit to two Ca(II) sites per subunit with low-millimolar K _ds. The variant scWT|E68A, in which metal ion binding in one subunit is abolished by mutation, also binds two Ca(II) ions in the WT subunit with low-millimolar K _ds. When there are no added metal ions, DNA binding affinity for scWT is tenfold stronger than that of the native WT, but tenfold weaker at saturating Ca(II) concentration. In the presence of Ca(II), scWT|E68A binds target DNA similarly to scWT, indicating that high-affinity substrate binding can be carried energetically by one metal-ion-binding subunit. Global analysis of DNA binding data for scWT|E68A suggests that the metal-ion-dependent behaviors observed for WT are reflective of independent subunit behavior. This characterization provides an understanding of subunit contributions in a homodimeric context.     

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles

Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimer’s disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that successfully silenced mutant tau or APP alleles. These plasmids should prove useful in experimental and therapeutic studies of Alzheimer’s disease. Our results suggest guiding principles for the production of allele-specific siRNA, and the general method described here should facilitate the production of gene-specific siRNAs.     

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications.     

Hunting behavior of Florida largemouth bass, Micropterus salmoides floridanus, in a channelized river

The distribution and behavior of Florida largemouth bass, Micropterus salmoides floridanus, and their main prey (sunfish, genus Lepomis, and the cichlid Tilapia mariae) were studied in southern Florida to determine how fish behave in the simplified habitats found in channelized rivers. Time budgets were constructed from focal animal observations on 69 bass. Patterns of behavior associated with hunting were performed during a significantly higher proportion of the time when bass were in vegetated habitats. Scan samples of the behavior of 236 observed bass revealed that hunting was more common in areas of high structural complexity. Only 38% of observed bass were solitary, with the majority occurring in groups with either conspecifics or in mixed-species groups with similar sized bluegill sunfish, Lepomis macrochirus. Largemouth bass (n=1014) and sunfish (n=1372) were significantly more abundant in areas with vegetation and were almost entirely absent from the water column in the center of the canal. All species of fish avoided the water column, where currents were swift and no cover was available. The structure of the habitat appears to be important in the way largemouth bass organize their activity patterns. This suggests that habitat availability in channelized rivers significantly influences important behaviors such as hunting, thus potentially altering energy budgets and population dynamics of both predator and prey.     

Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme

Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for beta cell function, suggesting a vital framework for the regulation of glucose homeostasis.     

Power to detect risk alleles using genome-wide tag SNP panels

Advances in high-throughput genotyping and the International HapMap Project have enabled association studies at the whole-genome level. We have constructed whole-genome genotyping panels of over 550,000 (HumanHap550) and 650,000 (HumanHap650Y) SNP loci by choosing tag SNPs from all populations genotyped by the International HapMap Project. These panels also contain additional SNP content in regions that have historically been overrepresented in diseases, such as nonsynonymous sites, the MHC region, copy number variant regions and mitochondrial DNA. We estimate that the tag SNP loci in these panels cover the majority of all common variation in the genome as measured by coverage of both all common HapMap SNPs and an independent set of SNPs derived from complete resequencing of genes obtained from SeattleSNPs. We also estimate that, given a sample size of 1,000 cases and 1,000 controls, these panels have the power to detect single disease loci of moderate risk (λ ~ 1.8–2.0). Relative risks as low as λ ~ 1.1–1.3 can be detected using 10,000 cases and 10,000 controls depending on the sample population and disease model. If multiple loci are involved, the power increases significantly to detect at least one locus such that relative risks 20%–35% lower can be detected with 80% power if between two and four independent loci are involved. Although our SNP selection was based on HapMap data, which is a subset of all common SNPs, these panels effectively capture the majority of all common variation and provide high power to detect risk alleles that are not represented in the HapMap data.