Targeting erythroblast-specific apoptosis in experimental anemia

Erythrocyte production is regulated by balancing precursor cell apoptosis and survival signaling. Previously, we found that BH3-only proapoptotic factor, Nix, opposed erythroblast-survival signaling by erythropoietin-induced Bcl-xl during normal erythrocyte formation. Since erythropoietin treatment of human anemia has limitations, we explored the therapeutic potential of abrogating Nix-mediated erythroblast apoptosis to enhance erythrocyte production. Nix gene ablation blunted the phenylhydrazine-induced fall in blood count, enhanced hematocrit recovery, and reduced erythroblast apoptosis, despite lower endogenous erythropoietin levels. Similar to erythropoietin, Nix ablation increased early splenic erythroblasts and circulating reticulocytes, while maintaining a pool of mature erythroblasts as erythropoietic reserve. Erythrocytes in Nix-deficient mice showed morphological abnormalities, suggesting that apoptosis during erythropoiesis not only controls red blood cell number, but also serves a “triage” function, preferentially eliminating abnormal erythrocytes. These results support the concept of targeting erythroblast apoptosis to maximize erythrocyte production in acute anemia, which may be of value in erythropoietin resistance. Abhinav Diwan and Andrew G. Koesters contributed equally to this work.     

The hide-and-seek game: men's perspectives on abortion and contraceptive use within marriage in a rural community in Zimbabwe

This paper is based on a study aimed at understanding the perceptions of men to induced abortion and contraceptive use within marriage in rural Zimbabwe. Two qualitative methods were combined. Men were found to view abortion not as a reproductive health problem for women. Instead, they described abortion as a sign of illicit sexual activity and contraceptive use as a strategy married women use to conceal their involvement in extramarital sexual activity. Men felt anxious and vulnerable for lack of control over women. In the absence of verbal communication on sexual matters, women and men resort to what are called here 'hide-and-seek' strategies, where women acquire and use contraceptives secretly while men search for evidence of such use. It is concluded that promoting women's sexual and reproductive health requires both short- and long-term strategies. The short-term strategy would entail providing women with reproductive technology they can use without risking violence. The long-term strategy would entail understanding men's concerns and the way these are manifested. In turn this requires the use of methodologies that encourage dialogue with research participants, in order to capture their deep meanings and experiences.     

Proapoptotic function of the MET tyrosine kinase receptor through caspase cleavage

The MET tyrosine kinase, the receptor of hepatocyte growth factor-scatter factor (HGF/SF), is known to be essential for normal development and cell survival. We report that stress stimuli induce the caspase-mediated cleavage of MET in physiological cellular targets, such as epithelial cells, embryonic hepatocytes, and cortical neurons. Cleavage occurs at aspartic residue 1000 within the SVD site of the juxtamembrane region, independently of the crucial docking tyrosine residues Y1001 or Y1347 and Y1354. This cleavage generates an intracellular 40-kDa MET fragment containing the kinase domain. The p40 MET fragment itself causes apoptosis of MDCK epithelial cells and embryonic cortical neurons, whereas its kinase-dead version is impaired in proapoptotic activity. Finally, HGF/SF treatment does not favor MET cleavage and apoptosis, confirming the known survival role of ligand-activated MET. Our results show that stress stimuli convert the MET survival receptor into a proapoptotic factor.     

Development of novel mixed-ligand oxotechnetium [SNS/S] complexes as potential 5-HT1A receptor imaging agents

The "3 + 1" ligand system [SN(R)S/S combination] was applied in order to synthesize neutral mixed-ligand oxotechnetium complexes of the general formula 99mTcO[SN(R)S]/[S] as potential 5-HT1A receptor imaging agents. The complexes are carrying the 1-(2-methoxyphenyl)piperazine moiety, a fragment of the true 5-HT1A antagonist WAY 100635, either on the monodentate ligand [S] or on the tridentate ligand [SN(R)S]. The complexes MO[EtN(CH2CH2S)2] [o-MeOC6H4N(CH2CH2)2NCH2CH2S] (3), MO[o- MeOC6H4N(CH2CH2)2N(CH2)3N(CH2CH2S)2][PhS] (6) and MO[o-MeOC6H4N(CH2CH2)2N(CH2)3N(CH2CH2S)2] [PhCH2CH2S] (9), where M = 99mTc, were prepared at tracer level using 99mTc glucoheptonate as precursor. For structural characterization, the analogous oxorhenium (M = Re, 1, 4 and 7, respectively) and oxotechnetium (M = 99gTc, 2, 5 and 8, respectively) complexes were prepared by ligand exchange reactions. All products were characterized by elemental analysis and spectroscopic methods. Complexes 1, 4 and 7 were further characterized by crystallographic analysis. For 1, the coordination geometry about rhenium can be described as trigonally distorted square pyramidal (tau = 0.36), while for 4 and 7, as distorted trigonal bipyramidal (tau = 0.66 and tau = 0.61, respectively). The coordination sphere about oxorhenium in all complexes is defined by the SNS donor atom set of the tridentate ligand and the sulfur atom of the monodentate coligand. The structure of the 99mTc complexes 3, 6 and 9 was established by comparative HPLC using authentic oxorhenium and oxotechnetium samples. The binding affinity of oxorhenium compounds for the 5-HT1A receptor subtype was determined in rat brain hippocampal preparations (IC50 = 6-31 nM). Preliminary tissue distribution data in healthy mice revealed the ability of all three 99mTc complexes to cross the intact blood-brain barrier (0.49-1.15% ID at 1 min p.i.). In addition, complexes 6 and 9 showed significant brain retention. These promising results have demonstrated that the SNS/S mixed-ligand system can be used in the development of 99mTc complexes as potential 5-HT1A receptor imaging agents.     

Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

Generation of skeletal muscles with forms adapted to their function is essential for normal movement. Muscle shape is patterned by the coordinated polarity of collectively migrating myoblasts. Constitutive inactivation of the protocadherin gene Fat1 uncoupled individual myoblast polarity within chains, altering the shape of selective groups of muscles in the shoulder and face. These shape abnormalities were followed by early onset regionalised muscle defects in adult Fat1-deficient mice. Tissue-specific ablation of Fat1 driven by Pax3-cre reproduced muscle shape defects in limb but not face muscles, indicating a cell-autonomous contribution of Fat1 in migrating muscle precursors. Strikingly, the topography of muscle abnormalities caused by Fat1 loss-of-function resembles that of human patients with facioscapulohumeral dystrophy (FSHD). FAT1 lies near the critical locus involved in causing FSHD, and Fat1 mutant mice also show retinal vasculopathy, mimicking another symptom of FSHD, and showed abnormal inner ear patterning, predictive of deafness, reminiscent of another burden of FSHD. Muscle-specific reduction of FAT1 expression and promoter silencing was observed in foetal FSHD1 cases. CGH array-based studies identified deletion polymorphisms within a putative regulatory enhancer of FAT1, predictive of tissue-specific depletion of FAT1 expression, which preferentially segregate with FSHD. Our study identifies FAT1 as a critical determinant of muscle form, misregulation of which associates with FSHD.     

Solid-phase synthesis,characterization and RNAi activity of branch and hyperbranch siRNAs

Linear, branch and hyperbranch siRNAs were effectively prepared for down-regulating GRP78 expression and inducing cell death in HepG2 liver cancer cells. Branch and hyperbranch GRP78 siRNAs were synthesized by automated solid-phase synthesis in good yields (44–78%) and isolated in excellent purities (>99%) following HPLC purification. Moreover, siRNAs adopted stable intramolecular hybrids as discerned by native PAGE and thermal denaturation studies. These sequences also exhibited the pre-requisite A-type helical trajectory for triggering RNAi activity as determined by CD spectroscopy. Biological studies confirmed potent suppression of GRP78 expression (50–60%) while compromising cancer cell viability by ～20%. Thus, branch and hyperbranch siRNAs may serve as potent siRNA candidates in cancer gene therapy applications.     

Immuno-localization of type-IV collagen in the blood-gas barrier and the epithelial–epithelial cell connections of the avian lung

The terminal respiratory units of the gas exchange tissue of the avian lung, the air capillaries (ACs) and the blood capillaries (BCs), are small and rigid: the basis of this mechanical feature has been highly contentious. Because the strength of the blood-gas barrier (BGB) of the mammalian lung has been attributed to the presence of type-IV collagen (T-IVc), localization of T-IVc in the basement membranes (BM) of the BGB and the epithelial–epithelial cell connections (E-ECCs) of the exchange tissue of the lung of the avian (chicken) lung was performed in order to determine whether it may likewise contribute to the strength of the BGB. T-IVc was localized in both the BM and the E-ECCs. As part of an integrated fibroskeletal scaffold on the lung, T-IVc may directly contribute to the strengths of the ACs and the BCs.     

Scaling metabolism from individuals to reef‐fish communities at broad spatial scales

Fishes contribute substantially to energy and nutrient fluxes in reef ecosystems, but quantifying these roles is challenging. Here, we do so by synthesising a large compilation of fish metabolic‐rate data with a comprehensive database on reef‐fish community abundance and biomass. Individual‐level analyses support predictions of Metabolic Theory after accounting for significant family‐level variation, and indicate that some tropical reef fishes may already be experiencing thermal regimes at or near their temperature optima. Community‐level analyses indicate that total estimated respiratory fluxes of reef‐fish communities increase on average ~2‐fold from 22 to 28 °C. Comparisons of estimated fluxes among trophic groups highlight striking differences in resource use by communities in different regions, perhaps partly reflecting distinct evolutionary histories, and support the hypothesis that piscivores receive substantial energy subsidies from outside reefs. Our study demonstrates one approach to synthesising individual‐ and community‐level data to establish broad‐scale trends in contributions of biota to ecosystem dynamics.