Ischemic preconditioning changes the pattern of coronary reactive hyperemia regardless of mitochondrial ATP-sensitive K(+) channel blockade

Ischemic preconditioning increases the velocity of vasodilatation and reduces the total hyperemic flow (THF) of a subsequent coronary reactive hyperemia (CRH). The increase in the velocity of vasodilatation has been shown to depend on an up-regulation of the endothelial release of nitric oxide, while the reduction of THF is attributed to an adenosine A(1) receptor-mediated mechanism. We investigated whether the changes in CRH induced by preconditioning ischemia (PI) can still be obtained after blockade of mitochondrial ATP-sensitive K(+) channels by sodium 5-hydroxydecanoate (5-HD), and whether the blockade per se affects the pattern of CRH.In anesthetized goats, flow was recorded from the left circumflex coronary artery (LCCA). CRH was obtained with the occlusion of LCCA for 15 s. PI was obtained by 2 cycles of 2.5 min of LCCA occlusion with a 5 min interval of reperfusion between the two occlusions. CRH was studied before and after i.v. administration of 5-HD (20 mg/kg), as well as in the presence of 5-HD after PI. Following 5-HD, the pattern of CRH remained unchanged. After 5-HD and PI, velocity of vasodilatation and total hyperemic flow of CRH showed the same changes as in previous studies after PI alone. It was concluded that the blockade of mitochondrial ATP-sensitive K(+) channels, which is reported to prevent myocardial protection, does not affect CRH and does not prevent PI from increasing the velocity of vasodilatation and reducing THF. These results demonstrate that the changes induced in CRH by preconditioning are independent of the opening of the mitochondrial ATP-sensitive K(+) channels.     

Transendothelial migratory pathways of V delta 1+TCR gamma delta+ and V delta 2+TCR gamma delta+ T lymphocytes from healthy donors and multiple sclerosis patients: involvement of phosphatidylinositol 3 kinase and calcium calmodulin-dependent kinase II

We have previously reported that the Vdelta2(+)TCRgammadelta(+) T lymphocyte subset, expressing the NK receptor protein 1a (NKRP1a; CD161), is expanded in patients with relapsing-remitting multiple sclerosis and uses this molecule to migrate through endothelium. In this work, we show that Vdelta1(+) and Vdelta2(+) gammadelta T lymphocytes use distinct signal transduction pathways to accomplish this function. Indeed, we have found that Vdelta1(+) cells lack NKRP1a and selectively express the platelet endothelial cell adhesion molecule 1 (PECAM1; CD31), which drives transendothelial migration of this cell subset, at variance with Vdelta2(+) T cells, which are PECAM1 negative and use NKRP1a for transmigration. Interestingly, when Vdelta2(+) T cells were pretreated with two specific inhibitors of the calcium calmodulin-dependent kinase II KN62 and KN93, but not with the inactive compound KN92, the number of migrating cells and the rate of transmigration were significantly decreased. In turn, the phosphatidylinositol 3 kinase blockers wortmannin and LY294002 exerted a dose-dependent inhibition of Vdelta1(+) cell migration. Finally, NKRP1a and PECAM1 engagement led to activation of different signal transduction pathways: indeed, oligomerization of NKRP1a on Vdelta2(+) T cells activates calcium calmodulin-dependent kinase II, while occupancy of PECAM1 on Vdelta1(+) cells triggers the phosphatidylinositol 3 kinase-dependent Akt/protein kinase Balpha activation. These findings suggest that subsets of gammadelta T lymphocytes may migrate to the site of lesion in multiple sclerosis using two different signaling pathways to extravasate.     

Activation tagging using the En-I maize transposon system in Arabidopsis

A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays) En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant called thread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that the En-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies.     

Effects of the aromatase inhibitor fadrozole on plasma sex steroid secretion,spermatogenesis and epididymis morphology in the lizard,Podarcis sicula

Recently, increasing importance has been attached to the role of estrogens and their receptors in male reproduction, since they have been found to be abundant in the male reproductive tract. In the lizard, Podarcis sicula, a seasonal breeder, estrogens seem to be involved in the regulation of testicular activity. Particularly, it has been hypothesized that the block of spermatogenesis and the complete regression of the epididymis and other secondary sexual characters (SSCs) in autumn might be due to high estrogen levels. To investigate the role of estrogens in the reproductive process of male lizards, we utilized Fadrozole ((AI) [4-(5,6,7,8-tetrahydroimidazole [1,5-a] pyridin-5-yl)-benzonitrile monohydrochloride] (CGS 16949A)), a nonsteroidal inhibitor of aromatase, the enzyme involved in the aromatization of androgens to estrogens, evaluating its effects on plasma sex-hormone release, spermatogenesis and epididymis morphology. For this purpose, adult male lizards, captured during the autumnal recrudescence, were intraperitoneally injected with 0.5 microg and 5 microg/g/body weight of AI for 15 and 30 days. In the animals treated with the higher AI dose, estrogen levels decreased if compared to the control groups, whereas androgen levels increased. Furthermore, histologic sections of testes and epididymes showed that the 30-day treatment with AI-induced spermatogenesis resumption with release of sperms into the large lumen of the seminiferous tubules, and the epididymes appeared more developed with moderately secreting columnar canal cells. Therefore, it is proposed that failure of spermatogenesis in autumn might be due to high estrogen levels.     

Structural and functional characterization of hBD-1(Ser35), a peptide deduced from a DEFB1 polymorphism

beta-Defensins are mammalian antimicrobial peptides that share a unique disulfide-bonding motif of six conserved cysteines. An intragenic polymorphism of the DEFB1 gene that changes a highly conserved Cys to Ser in the peptide coding region has recently been described. The deduced peptide cannot form three disulfide bonds, as one of the cysteines is unpaired. We have determined the cysteine connectivities of a corresponding synthetic hBD-1(Ser35) peptide, investigated the structure by circular dichroism spectroscopy, and assayed the in vitro antimicrobial activity. Despite a different arrangement of the disulfides, hBD-1(Ser35) proved as active as hBD-1 against the microorganisms tested. This activity likely depends on the ability of hBD-1(Ser35) to adopt an amphipathic conformation in hydrophobic environment, similar to the wild type peptide, as suggested by CD spectroscopy.     

Modulation of the expression of GABA(A) receptors in rat cerebellar granule cells by protein tyrosine kinases and protein kinase C

The expression of GABA(A) receptors in rat cerebellar granules in culture has been studied by beta(2/3) subunit immunocytochemistry and fluorescence confocal microscopy. These cells show labeling all over the cell bodies' plasma membrane and dendrites. Treatment with the protein tyrosine kinase (PTK) inhibitor genistein results in a decrease of the labeling associated with the beta(2/3) subunit in both cell bodies and dendrites. No effect was found with an inactive genistein analogue, daidzein. A similar effect was found with a protein kinase C (PKC) activator, phorbol myristate acetate (PMA). The effects of genistein and PMA are additive.The interpretation of the results is that PTK inhibition blocks exocytotic deposit of newly synthesized GABA(A) receptors onto the neuronal plasma membrane. On the other hand, PKC activation speeds up endocytotic removal of GABA(A) receptors.     

MADS-box protein complexes control carpel and ovule development in Arabidopsis

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.     

Carbohydrate and amino acid metabolism in Tuber borchii mycelium during glucose utilization: a (13)C NMR study

The metabolism of [1-13C]glucose in the vegetative mycelium of the ectomycorrhizal ascomycete Tuber borchii was studied in order to characterize the biochemical pathways for the assimilation of glucose and amino acid biosynthesis. The pathways were characterized using nuclear magnetic resonance spectroscopy in conjunction with [1-13C]glucose labeling. The enzymes of mannitol cycle and ammonium assimilation were also evaluated. The majority of the 13C label was incorporated into mannitol and this polyol was formed via a direct route from absorbed glucose. Amino acid biosynthesis was also an important sink of assimilated carbon and 13C was mainly incorporated into alanine and glutamate. From this intramolecular 13C enrichment, it is concluded that pyruvate, arising from [1-13C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase and pyruvate carboxylase before entering the Krebs cycle. The transfer of 13C-labeled mycelium on [12C]glucose showed that mannitol, alanine, and glutamate carbon were used to synthesize glutamine and arginine that likely play a storage role.     

Cancer therapy: switching off oncogenes

Cancer derives from a cell clone that has accumulated genetic and epigenetic changes that influence its phenotype and finally enable it to escape from the normal controls of proliferation. A recent paper shows that, in myc-induced tumours, the inactivation of this oncogene produces the regression of the tumours and the differentiation of the tumour cells into mature osteocytes.1 In addition, a further reactivation of myc in these cells does not restore the malignant phenotype but induces apoptosis. This discovery could lead to an innovative therapeutic strategy.