Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein- 1 alpha

BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU-S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G- CSF) and the combination of G-CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100- fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117-, and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB- 10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation.     

The inability of isoproterenol or propranolol to alter the lateral dimensions of experimentally induced myocardial infarcts

Summary The relationship between the blood flow pattern immediately following coronary artery occlusion and the resulting infarct 24 hours later was studied in dogs treated with isoproterenol (0.5 g/kg/min for 2 hours) or with propranolol (2mg/kg every 6 hours). The coronary artery of a closed chest dog was perfused via a special cannula with arterial blood. A 2-mm diameter plastic bead was introduced into the perfusate to embolize a coronary branch. One minute after occlusion, radiolabelled microspheres were injected into the perfusate. The dogs were then allowed to recover. 24 hours later the dogs were reanesthetized and their hearts removed. The hearts were sliced into 4 mm thick sections and the microsphere distribution was visualized by autoradiography of the tissue. Superimposition of developed autoradiographs and tracings of the infarct pattern of stained sections allowed direct comparison of the blood flow pattern immediately after occlusion to the eventual pattern of infarction. In all 8 control dogs, all 6 isoproterenol dogs and all 12 propranolol dogs the lateral borders of blood flow and infarction were superimposable indicating no lateral change in infarct size resulting from treatment. In the control group there was a subepicardial region of the ischemic zone which did not infarct (15.2±2.3% of the ischemic zone). Though isoproterenol did not significantly change the size of this zone, propranolol increased it to 35.9±6,5% (p<0.005) indicating vertical but not lateral salvage.Supported by Grant HL-20648 from NIH: HLBI and a Grant-in-aid from the American Heart Association and with funds contributed in part by the Northwest Ohio Chapter, Inc.     

Comparative analysis of mammalian Y chromosomes illuminates ancestral structure and lineage-specific evolution

Although more than thirty mammalian genomes have been sequenced to draft quality, very few of these include the Y chromosome. This has limited our understanding of the evolutionary dynamics of gene persistence and loss, our ability to identify conserved regulatory elements, as well our knowledge of the extent to which different types of selection act to maintain genes within this unique genomic environment. Here, we present the first MSY (male-specific region of the Y chromosome) sequences from two carnivores, the domestic dog and cat. By combining these with other available MSY data, our multiordinal comparison allows for the first accounting of levels of selection constraining the evolution of eutherian Y chromosomes. Despite gene gain and loss across the phylogeny, we show the eutherian ancestor retained a core set of 17 MSY genes, most being constrained by negative selection for nearly 100 million years. The X-degenerate and ampliconic gene classes are partitioned into distinct chromosomal domains in most mammals, but were radically restructured on the human lineage. We identified multiple conserved noncoding elements that potentially regulate eutherian MSY genes. The acquisition of novel ampliconic gene families was accompanied by signatures of positive selection and has differentially impacted the degeneration and expansion of MSY gene repertoires in different species.Y chromosomes have arisen independently in divergent evolutionary lineages across the eukaryotic tree of life (Rice 1996; Marin and Baker 1998; Liu et al. 2004; Graves 2006; Koerich et al. 2008; Carvalho et al. 2009; Kaiser and Bachtrog 2010). While many genes are known to be Y-linked, the actual number of Y chromosomes that have been sequenced is extremely small (Skaletsky et al. 2003; Hughes et al. 2005; Kuroki et al. 2006; Clark et al. 2007; Koerich et al. 2008; Carvalho et al. 2009; Hughes et al. 2010, 2012) in relation to the rapid rate at which whole genomes are presently being sequenced. This reduced emphasis on sequencing Y chromosomes can be primarily attributed to their presumed low gene content and large amounts of repetitive DNA, that is often arrayed into long stretches of nearly identical sequence which precludes the use of shotgun sequencing approaches to assemble Y chromosome sequence into large, contiguous scaffolds. These assembly problems are further exacerbated when applying short-read next-generation sequence methods (Alkan et al. 2011).The most completely sequenced and annotated Y chromosomes are from three recently diverged catarrhine primates: human, chimpanzee, and rhesus macaque (Skaletsky et al. 2003; Hughes et al. 2005, 2010, 2012; Kuroki et al. 2006 ). Comparisons between these species offer an important glimpse into the immense structural variation and complexity that can emerge on the Y within a very short period of evolutionary time. However, these three primate species last shared a common ancestor ∼21 million years ago (Mya) (Meredith et al. 2011) and thus offer limited comparative breadth to discriminate characteristics present across most mammalian Y chromosomes from idiosyncratic features reflective of a small evolutionary sample. This lack of phylogenetic scope has (1) hampered identification of the ancestral properties of eutherian Y chromosomes, (2) obscured broader patterns of evolutionary constraint and selection in a nonrecombining environment, and (3) hidden the frequency with which novel genes arise and/or acquire new functions in species with diverse phenotypes and reproductive strategies.To expand our knowledge of eutherian Y chromosome structure and gene function, we generated the first extensive MSY (male-specific Y chromosome) sequence from two members of the Carnivora: the domestic cat, Felis silvestris catus, and domestic dog, Canis lupus familiaris. These two carnivore genomes provide a phylogenetically distinct vantage point with which to interpret the evolutionary patterns observed in the primate MSY comparisons, diverging from each other ∼55 Mya, and from primates ∼92 Mya (Meredith et al. 2011). Our combined analysis of two carnivore and three primate MSY sequences, together with physical mapping and functional sequence data from the mouse MSY, represent the first multiordinal comparison assessing deeper levels of evolutionary constraint. We also present a complete analysis of patterns of negative and positive selection to assess their effects on MSY degeneration and expansion.     

\dot{V}_{{{\text{O}}_{2} { \max }}} is not altered by self-pacing during incremental exercise

We tested the hypothesis that incremental cycling to exhaustion that is paced using clamps of the rating of perceived exertion (RPE) elicits higher $ \dot{V}_{{{\text{O}}_{2} { \max }}} $ values compared to a conventional ramp incremental protocol when test duration is matched. Seven males completed three incremental tests to exhaustion to measure $ \dot{V}_{{{\text{O}}_{2} { \max }}} $ . The incremental protocols were of similar duration and included: a ramp test at 30 W min^?1 with constant cadence (RAMP1); a ramp test at 30 W min^?1 with cadence free to fluctuate according to subject preference (RAMP2); and a self-paced incremental test in which the power output was selected by the subject according to prescribed increments in RPE (SPT). The subjects also completed a $ \dot{V}_{{{\text{O}}_{2} { \max }}} $ ‘verification’ test at a fixed high-intensity power output and a 3-min all-out test. No difference was found for $ \dot{V}_{{{\text{O}}_{2} { \max }}} $ between the incremental protocols (RAMP1 = 4.33 ± 0.60 L min^?1; RAMP2 = 4.31 ± 0.62 L min^?1; SPT = 4.36 ± 0.59 L min^?1; P > 0.05) nor between the incremental protocols and the peak $ \dot{V}_{{{\text{O}}_{2} }} $ measured during the 3-min all-out test (4.33 ± 0.68 L min^?1) or the $ \dot{V}_{{{\text{O}}_{2} { \max }}} $ measured in the verification test (4.32 ± 0.69 L min^?1). The integrated electromyogram, blood lactate concentration, heart rate and minute ventilation at exhaustion were not different (P > 0.05) between the incremental protocols. In conclusion, when test duration is matched, SPT does not elicit a higher $ \dot{V}_{{{\text{O}}_{2} { \max }}} $ compared to conventional incremental protocols. The striking similarity of $ \dot{V}_{{{\text{O}}_{2} { \max }}} $ measured across an array of exercise protocols indicates that there are physiological limits to the attainment of $ \dot{V}_{{{\text{O}}_{2} { \max }}} $ that cannot be exceeded by self-pacing.