Nuclear transfer and iPS may work best together

A short meeting, held as an Arthur Sackler Colloquium of the United States National Academy of Sciences, was organized by Douglas Wallace, Susan Bryant, and Peter Donovan under the heading "Therapeutic Cloning: Where Do We Go from Here?" on October 8 and 9, 2007. The individual components required for therapeutic cloning now exist. The question, therefore, is what constraints presently limit or prevent its application to human therapy.     

Production of low molecular weight chemoattractants for leukocytes by periovulatory ovine follicles

Studies were performed to determine if periovulatory ovine follicles secrete chemoattractants for leukocytes, and if so, to begin to elucidate the chemical nature of such factors. Tissues were obtained at 0, 12, 24, and 36 h after initiation of the preovulatory surge of luteinizing hormone and placed in short-term incubation (ovulation occurs at approximately 24 h). Follicular-conditioned medium was tested for its ability to attract leukocytes by utilizing a linear under-agarose assay: chemotaxis was quantified as a function of the leading front of migration of cells. Neutrophils and eosinophils were attracted toward media conditioned with tissues of 24 and 36 h. Monocytes responded toward medium of tissues collected at 36 h. There was no evidence for chemoattraction of basophils or lymphocytes. Chemoattractant activity for granulocytes and monocytes was of low molecular weight origin (less than 3000) and water-soluble. High-performance liquid chromatographic separation of this sample produced a distinct peak with recoverable activity. The isolated fraction was rich in glycine. Eosinophils also migrated toward an additional low molecular weight attractant that was extracted into ethyl acetate. Leukocytes attracted into periovulatory follicles might produce substances (eg., proteolytic enzymes and angiogenic factors) that play a role in the mechanisms of ovulation and luteinization.     

Sequence analysis of leukocyte chemoattractant peptides secreted by periovulatory ovine follicles.

Cells of the white blood series infiltrate ovarian follicles during the ovulatory process. A segment of the wall of periovulatory ovine follicles was incubated and conditioned media subjected to ultrafiltration. Leukocyte chemoattractant activity of media was measured using a linear under-agarose migration assay. Bioactivity was recovered following filtration through a 3000 molecular weight cut-off membrane. Filtrate was then fractionated by reverse phase high performance liquid chromatography. Peptides recovered from two fractions with significant chemoattractant activity were sequenced. One fraction contained 16 amino acid residues with repeating triplets of Gly-X-Y, where X and Y were often proline and hydroxyproline, respectively. Because this motif is characteristic of alpha collagens, and since thecal collagen is degraded during the mechanics of ovulation, it appears that this chemoattractant is derived from the connective tissue matrix of the follicle. Peptide isolated from the other bioactive chromatographic fraction was 15 amino acids in length, and rich in glycine, but did not contain imino acids. To our knowledge this is the first report of purification of leukocyte chemoattractants of reproductive tissue origin. Resident follicular granulocytes and mononuclear cells are capable of secreting a broad spectrum of potent chemicals that could be involved in the mechanisms of ovulation and luteinization.     

Novel flow cytometric approach for the detection of adipocyte subpopulations during adipogenesis

The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified a subpopulation of adipose-derived stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of adipose-derived stromal cells that express CD36^{intermediate/high} during adipocyte differentiation in vitro. The gradual increase of CD36^{intermediate/high/}NR^positive cells during the 21 day adipogenesis induction period correlated with upregulation of adipogenesis-associated gene expression.     

Scale-dependent spatial variability of coral assemblages along the Florida Reef Tract

 Coral reef communities of the western Atlantic have changed over the past two to three decades, but the magnitude and causes of this change remain controversial. Part of the problem is that small-scale patterns observed on individual reefs have been erroneously extrapolated to landscape and geographic scales. Understanding how reef coral assemblages vary through space is an essential prerequisite to devising sampling strategies to track the dynamics of coral reefs through time. In this paper we quantify variation in the cover of hard corals in spur-and-groove habitats (13–19 m depth) at spatial scales spanning five orders of magnitude along the Florida Reef Tract. A videographic sampling program was conducted to estimate variances in coral cover at the following hierarchical levels and corresponding spatial scales: (1) among transects within sites (0.01- to 0.1-km scale), (2) among sites within reefs (0.5- to 2-km scale), (3) among reefs within sectors of the reef tract (10- to 20-km scale), and (4) among sectors of the reef tract (50- to 100-km scale). Coral cover displayed low variability among transects within sites and among sites within reefs. This means that transects from a site adequately represented the variability of the spur-and-groove habitat of the reef as a whole. Variability among reefs within sectors was highly significant, compared with marginally significant variability among sectors. Estimates from an individual reef, therefore, did not adequately characterize nearby reefs, nor did those estimates sufficiently represent variability at the scale of the sector. The structure and composition of coral reef communities is probably determined by the interaction of multiple forcing functions operating on a variety of scales. Hierarchical analyses of coral assemblages from other geographic locations have detected high variability at scales different from those in the present study. A multiscale analysis should, therefore, precede any management decisions regarding large reef systems such as the Florida Reef Tract. Accepted: 19 July 1999     

Ndel1 alters its conformation by sequestering cAMP-specific phosphodiesterase-4D3 (PDE4D3) in a manner that is dynamically regulated through Protein Kinase A (PKA)

The involvement of the Nuclear distribution element-like (Ndel1; Nudel) protein in the recruitment of the dynein complex is critical for neurodevelopment and potentially important for neuronal disease states. The PDE4 family of phosphodiesterases specifically degrades cAMP, an important second messenger implicated in learning and memory functions. Here we show for the first time that Ndel1 can interact directly with PDE4 family members and that the interaction of Ndel1 with the PDE4D3 isoform is uniquely disrupted by elevation of intracellular cAMP levels. While all long PDE4 isoforms are subject to stimulatory PKA phosphorylation within their conserved regulatory UCR1 domain, specificity for release of PDE4D3 is conferred due to the PKA-dependent phosphorylation of Ser13 within the isoform-specific, unique amino-terminal domain of PDE4D3. Scanning peptide array analyses identify a common region on Ndel1 for PDE4 binding and an additional region that is unique to PDE4D3. The common site lies within the stutter region that links the second coiled-coil region to the unstable third coiled-coil regions of Ndel1. The additional binding region unique to PDE4D3 penetrates into the start of the third coiled-coil region that can undergo tail-to-tail interactions between Ndel1 dimers to form a 4 helix bundle. We demonstrate Ndel1 self-interaction in living cells using a BRET approach with luciferase- and GFP-tagged forms of Ndel1. BRET assessed Ndel1–Ndel1 self-interaction is amplified through the binding of PDE4 isoforms. For PDE4D3 this effect is ablated upon elevation of intracellular cAMP due to PKA-mediated phosphorylation at Ser13, while the potentiating effects of PDE4B1 and PDE4D5 are resistant to cAMP elevation. PDE4D long isoforms and Ndel1 show a similar sub-cellular distribution in hippocampus and cortex and locate to post-synaptic densities. We show that Ndel1 sequesters EPAC, but not PKA, in order to form a cAMP signalling complex. We propose that a key function of the Ndel1 signalling scaffold is to signal through cAMP by sequestering EPAC, whose activity may thus be specifically regulated by sequestered PDE4 that also stabilizes Ndel1–Ndel1 self-interaction. In the case of PDE4D3, its association with Ndel1 is dynamically regulated by PKA input through its ability to phosphorylate Ser13 in the unique N-terminal region of this isoform, triggering the specific release of PDE4D3 from Ndel1 when cAMP levels are elevated. We propose that Ser13 may act as a redistribution trigger in PDE4D3, allowing it to dynamically re-shape cAMP gradients in distinct intracellular locales upon its phosphorylation by PKA.