Expression of constitutively active guanylate cyclase in cardiomyocytes inhibits the hypertrophic effects of isoproterenol and aortic constriction on mouse hearts

Evidence from several rodent models has suggested that a reduction of either atrial natriuretic peptide or its receptor in the heart affects cardiac remodeling by promoting the onset of cardiac hypertrophy. The atrial natriuretic peptide receptor mediates signaling at least in part via the generation of intracellular cyclic GMP. To directly test whether accumulation of intracellular cyclic GMP conveys protection against cardiac hypertrophy, we engineered transgenic mice that overexpress a catalytic fragment of constitutively active guanylate cyclase domain of the atrial natriuretic peptide receptor in a cardiomyocyte-specific manner. Expression of the transgene increased the intracellular concentration of cyclic GMP specifically within cardiomyocytes and had no detectable effect on cardiac performance under basal conditions. However, expression of the transgene attenuated the effects of the pharmacologic hypertrophic agent isoproterenol on cardiac wall thickness and prevented the onset of the fetal gene expression program normally associated with cardiac hypertrophy. Likewise, expression of the transgene inhibited the hypertrophic effects of abdominal aortic constriction, since it abolished its effects on ventricular wall thickness and greatly attenuated its effects on cardiomyocyte size. Altogether, our results suggest that cyclic GMP is a cardioprotective agent against hypertrophy that acts via a direct local effect on cardiomyocytes.     

Oxygen deficiency affects carbohydrate reserves in overwintering forage crops

Anaerobic conditions developing under an ice cover affect winter survival and spring regrowth of economically important perennial crops. The objective was to compare, during a prolonged period of low (<2%) O2 at low temperature, the concentration of carbohydrates of four plant species contrasting in their resistance to oxygen deficiency. Four perennial forage species, lucerne (Medicago sativa L.), red clover (Trifolium pratense L.), timothy (Phleum pratense L.), and cocksfoot (Dactylis glomerata L.) were subjected to a progressively developing oxygen deficiency stress by enclosing potted plants in gas-tight bags in late autumn for overwintering in an unheated greenhouse. Timothy was previously reported to be more resistant to oxygen deficiency than the three other species. Non-structural carbohydrates increased and remained at a higher concentration in timothy than in the other three species under low O2 concentration. Concentrations of sucrose, fructose, glucose, and fructans increased in response to oxygen deficiency in timothy, whereas the concentration of soluble sugars decreased under the same conditions in lucerne, red clover, and cocksfoot. The gene expression of glyceraldehyde-3-phosphate dehydrogenase increased in response to low oxygen concentration in oxygen deficiency-sensitive lucerne while it remained unchanged in the oxygen deficiency-resistant timothy. It is concluded that timothy maintains higher carbohydrate reserves under oxygen deficiency, a specific feature that could favour its winter survival and spring regrowth.     

Fast renal trapping of porcine Luteinizing Hormone (pLH) shown by <Superscript>123</Superscript>I-scintigraphic imaging in rats explains its short circulatory half-life

Background  

Sugar moieties of gonadotropins play no primary role in receptor binding but they strongly affect their circulatory half-life and consequently their in vivo biopotencies. In order to relate more precisely hepatic trapping of these glycoproteic hormones with their circulatory half-life, we undertook a comparative study of the distribution and elimination of porcine LH (pLH) and equine CG (eCG) which exhibit respectively a short and a long half-life. This was done first by following half-lives of pLH in piglets with hepatic portal circulation shunted or not. It was expected that such a shunt would enhance the short half-life of pLH. Subsequently, scintigraphic imaging of both ¹²³I-pLH and ¹²³I-eCG was performed in intact rats to compare their routes and rates of distribution and elimination.     

Effect of prooxidant agents added at the morula/blastocyst stage on bovine embryo development,cell death and glutathione content

Two prooxidant agents, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), a generator of free radicals in the culture medium, and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, were used to reinforce from the morula stage (day 5 post-insemination, p.i.) the oxidative stress encountered by bovine embryos in culture. Exposure to increasing concentrations of both prooxidants from the morula stage did not affect blastocyst formation but some blastocysts were found degenerated on day 8 in a dose-dependent manner (0, 0.001, 0.01, 0.1 mM AAPH gave respectively 0, 10%, 32%, 48% degeneration, while 0, 0.1, 0.2, 0.4 mM BSO led respectively to 0, 14%, 30%, 41% degeneration). Hatching rates and cell numbers of surviving blastocysts were not affected. Morulae and early blastocysts exposed from day 5 to day 6 p.i. appeared more resistant than expanded blastocysts (75-80% survival vs 20-65%; p < 0.05). Treatment with BSO significantly decreased the level of reduced glutathione in day 7 blastocysts (0.02 vs 0.42 pmol per embryo in the control) while AAPH had no effect (0.38 pmol per embryo). The proportion ofcells showing membrane lesions was increased in degenerated blastocysts from day 7.5 p.i. In AAPH-treated, but not in BSO-treated embryos, cell membrane permeabilisation seems to occur before blastocyst degeneration. DNA fragmentation evaluated by the TUNEL technique was increased in day 7 blastocysts by both prooxidants (2.8 +/- 0.4 in the control group vs 4.5 +/- 0.4 and 6.0 +/- 0.4 respectively in the AAPH- and BSO-treated groups). Addition of an inhibitor of caspase-3, DEVD-CHO, partially prevented DNA fragmentation, indicating that prooxidant treatment induced a caspase-dependent pathway of apoptosis.     

Deletion of cytosolic phospholipase A2 promotes striated muscle growth

Generation of arachidonic acid by the ubiquitously expressed cytosolic phospholipase A2 (PLA2) has a fundamental role in the regulation of cellular homeostasis, inflammation and tumorigenesis. Here we report that cytosolic PLA2 is a negative regulator of growth, specifically of striated muscle. We find that normal growth of skeletal muscle, as well as normal and pathologic stress-induced hypertrophic growth of the heart, are exaggerated in Pla2g4a-/- mice, which lack the gene encoding cytosolic PLA2. The mechanism underlying this phenotype is that cytosolic PLA2 negatively regulates insulin-like growth factor (IGF)-1 signaling. Absence of cytosolic PLA2 leads to sustained activation of the IGF-1 pathway, which results from the failure of 3-phosphoinositide-dependent protein kinase (PDK)-1 to recruit and phosphorylate protein kinase C (PKC)-zeta, a negative regulator of IGF-1 signaling. Arachidonic acid restores activation of PKC-zeta, correcting the exaggerated IGF-1 signaling. These results indicate that cytosolic PLA2 and arachidonic acid regulate striated muscle growth by modulating multiple growth-regulatory pathways.     

Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening

New opportunities in mammalian functional genomics are emerging through the combination of high throughput technology and methods that allow manipulation of gene expression in living cells. Here we describe the application of an RNAi-based forward genomics approach toward understanding the biology and mechanism of TRAIL-induced apoptosis. TRAIL is a TNF superfamily member that induces selective cytotoxicity of tumor cells when bound to its cognate receptors. In addition to detecting well-characterized genes in the apoptosis pathway, we uncover several modulators including DOBI, a gene required for progression of the apoptotic signal through the intrinsic mitochondrial cell death pathway, and MIRSA, a gene that acts to limit TRAIL-induced apoptosis. Moreover, our data suggest a role for MYC and the WNT pathway in maintaining susceptibility to TRAIL. Collectively, these observations offer several insights on how TRAIL mediates the selective killing of tumor cells and demonstrate the utility of large-scale RNAi screens in mammalian cells.     

Distribution of the intermediate filament nestin in the muscularis propria of the human gastrointestinal tract

The intermediate filament nestin is expressed in neural stem cells, neuroectodermal tumors and various adult tissues. In the gastrointestinal (GI) tract, nestin has been reported in glial cells. Recently, nestin has been reported in interstitial cells of Cajal (ICC) and in gastrointestinal stromal tumors, thought to derive from ICC. Here we investigated nestin immunoreactivity (-ir) in the normal human GI tract, with emphasis on Kit-ir ICC. Two different antibodies specific for human nestin and multicolor high-resolution confocal microscopy were used on material from our human GI tissue collection. The staining pattern of both nestin antibodies was similar. In labeled cells, nestin-ir appeared filamentous. Most intramuscular ICC in antrum and all myenteric ICC (ICC-MP) in small intestine were nestin-ir, while nestin-ir was not detected in deep muscular plexus ICC. In the colon, some - but not all - ICC-MP and most ICC in the circular musculature were nestin-ir while nestin-ir was not detected in ICC in the longitudinal musculature and in the submuscular plexus. In addition, many Kit-negative cells were nestin-ir in all regions. Neurons and smooth muscle cells were consistently nestin negative, while most S100-ir glial cells were nestin-ir. In addition, nestin-ir was also present in some CD34-ir fibroblast-like cells, in endothelium and in other cell types in the mucosa and serosa. In conclusion, nestin-ir is abundantly present in the normal human GI tract. Among a number of cell types, several, but not all, subpopulations of Kit-ir ICC were nestin-ir. The functional significance of nestin in the GI tract remains obscure.     

Maintenance of double-stranded telomeric repeats as the critical determinant for cell viability in yeast cells lacking Ku

The Saccharomyces cerevisiae Ku complex, while important for nonhomologous DNA end joining, is also necessary for maintaining wild-type telomere length and a normal chromosomal DNA end structure. Yeast cells lacking Ku can grow at 23 degrees C but are unable to do so at elevated temperatures due to an activation of DNA damage checkpoints. To gain insights into the mechanisms affected by temperature in such strains, we isolated and characterized a new allele of the YKU70 gene, yku70-30(ts). By several criteria, the Yku70-30p protein is functional at 23 degrees C and nonfunctional at 37 degrees C. The analyses of telomeric repeat maintenance as well as the terminal DNA end structure in strains harboring this allele alone or in strains with a combination of other mutations affecting telomere maintenance show that the altered DNA end structure in yeast cells lacking Ku is not generated in a telomerase-dependent fashion. Moreover, the single-stranded G-rich DNA on such telomeres is not detected by DNA damage checkpoints to arrest cell growth, provided that there are sufficient double-stranded telomeric repeats present. The results also demonstrate that mutations in genes negatively affecting G-strand synthesis (e.g., RIF1) or C-strand synthesis (e.g., the DNA polymerase alpha gene) allow for the maintenance of longer telomeric repeat tracts in cells lacking Ku. Finally, extending telomeric repeat tracts in such cells at least temporarily suppresses checkpoint activation and growth defects at higher temperatures. Thus, we hypothesize that an aspect of the coordinated synthesis of double-stranded telomeric repeats is sensitive to elevated temperatures.