Expression and localization of caveolins during postnatal development in rat heart: implication of thyroid hormone.

Caveolins modulate signaling pathways involved in cardiac development. Caveolin-1 exists in two isoforms: the beta-isoform derivates from an alternative translational start site that creates a protein truncated by 31 amino acids, mainly expressed in endothelial cells, whereas caveolin-3 is present in muscle cells. Our aim was to define caveolin distribution and expression during cardiac postnatal development using immunofluorescence and Western blotting. Caveolin-3 sarcolemmal labeling appeared as dotted lines from days 1 to 5 and as continuous lines after 14 days of age. Caveolin-3 expression, low at birth, increased (4-fold) to reach a maximum (P < 0.05) by day 5 and then decreased to stabilize in adults. Total caveolin-1 and its alpha-isoform were codistributed at birth in endothelial and smooth muscle cells; afterward, only the caveolin-1alpha labeling became limited to endothelium. Quantitative analysis indicated a similar temporal pattern of both total caveolin-1 and caveolin-1alpha expression, suggesting that caveolin-1alpha and -1beta are coregulated; the caveolin-1alpha levels increased fourfold by day 5 to reach a maximum by day 14 (P < 0.05). Tyrosine-14-caveolin-1 phosphorylation, low at birth, increased suddenly around day 14 (8-fold vs. day 1) and returning afterward to basal level. Because the T3/T4 level is maximal by day 14, caveolin-1 expression/phosphorylation profiles were analyzed in hypothyroid heart. The levels of caveolin-1alpha and consequently tyrosine-14-caveolin-1 phosphorylation, but not that of caveolin-3, decreased (50%) in hypothyroid 14-day-old rats. Our data demonstrate that, during postnatal cardiac growth, 1) caveolins are distinctly regulated, and 2) thyroid hormones are involved in caveolin-1alpha expression.     

Comparative study of storage compound breakdown in germinating seeds of three lupine species

Storage lipid and protein breakdown in germinating seeds of yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupine (L. mutabilis Sweet) and regulatory function of sucrose were investigated. Less oil bodies were detected in organs of yellow lupine seeds, whereas the highest content of oil bodies was noticed in the Andean lupine seeds. Mature, air-dried yellow, white and Andean lupine seeds do not contain starch. Starch grains appear the earliest in white lupine seeds during imbibition. Sucrose deficiency in tissues enhances breakdown of storage lipid, protein and temporary starch in cotyledons. In sucrose starved embryo axes of all investigated lupine species, an increased level of vacuolization was noted. Interconnections between catabolism of storage protein and storage lipid in germinating lupine seeds were identified by applying ¹⁴C-acetate. To assess the importance of key processes in storage lipid breakdown NaF (inhibitor of glycolysis and gluconeogenesis), KCN, NaN₃ and SHAM (inhibitors of mitochondrial electron transport chain) and MSO (inhibitor of glutamine synthetase) were used. Radioactivity coming from ¹⁴C-acetate was released as ¹⁴CO₂ but mostly was incorporated into ethanol-soluble fraction of embryo axes and cotyledons. Respiratory inhibitors caused a significant decrease in ¹⁴CO₂ and ethanol fractions in all three lupine species studied. MSO stimulated release of ¹⁴CO₂ and radioactivity of ethanol fractions in yellow lupine organs fed with sucrose, but in Andean lupine MSO enhanced the production of ¹⁴CO₂ and radioactivity of ethanol fractions both in organs fed and not fed with sucrose. Different strategies of storage compound breakdown are proposed, depending on relative proportion in storage protein and lipid content in lupine seeds.     

Proteasome inhibitors impair RANKL‐induced NF‐κB activity in osteoclast‐like cells via disruption of p62, TRAF6, CYLD,and IκBα signaling cascades

Proteasome inhibitors represent a promising therapy for the treatment of relapsed and/or refractory multiple myeloma, a disease that is concomitant with osteolysis and enhanced osteoclast formation. While blockade of the proteosome pathway has been recently shown to influence osteoclast formation and function, the precise molecular cascade underlying these effects is presently unclear. Here, we provide evidence that proteasome inhibitors directly impair osteoclast formation and function via the disruption of key RANK‐mediated signaling cascades. Disruption of the proteosome pathway using selective inhibitors (MG‐132, MG‐115, and epoxomicin) resulted in the accumulation of p62 and CYLD, and altered the subcellular targeting and distribution of p62 and TRAF6 in osteoclast‐like cells. Proteosome inhibition also blocked RANKL‐induced NF‐κB activation, IκBα degradation and nuclear translocation of p65. The disruption in RANK‐signaling correlated dose‐dependently with an impairment in osteoclastogenesis, with relative potency epoxomicin > MG‐132 > MG‐115 based on equimolar concentrations. In addition, these inhibitors were found to impact osteoclastic microtubule organization and attenuate bone resorption. Based on these data we propose that deregulation of key RANK‐mediated signaling cascades (p62, TRAF6, CYLD, and IκBα) underscores proteasome‐mediated inhibition of osteolytic bone conditions. J. Cell. Physiol. 220: 450–459, 2009. © 2009 Wiley‐Liss, Inc.     

Phenotypic subunit composition of the tobacco (Nicotiana tabacum L.) vacuolar-type H-translocating ATPase

The model plant tobacco (Nicotiana tabacum L.) was chosen for a survey of the subunit composition of the V-ATPase at the protein level. V-ATPase was purified from tobacco leaf cell tonoplasts by solubilization with the nonionic detergent Triton X-100 and immunoprecipitation. In the purified fraction 12 proteins were present. By matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and amino acid sequencing 11 of these polypeptides could be identified as subunits A, B, C, D, F, G, c, d and three different isoforms of subunit E. The polypeptide which could not be identified by MALDI analysis might represent subunit H. The data presented here, for the first time, enable an unequivocal identification of V-ATPase subunits after gel electrophoresis and open the possibility to assign changes in polypeptide composition to variations in respective V-ATPase subunits occurring as a response to environmental conditions or during plant development.     

Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa.

The Chenopodiaceae Suaeda salsa L. was grown under different salt concentrations and under osmotic stress. The fresh weight was markedly stimulated by 0.1 M NaCl, 0.4 M NaCl and 0.1 M KCl and reduced by osmotic stress (PEG iso-osmotic to 0.1 M NaCl). Treatment with 0.4 M KCl severely damaged the plants. Membrane vesicle fractions containing tonoplast vesicles were isolated by sucrose gradient from leaves of the S. salsa plants and modulations of V-ATPase and V-PPase depending on the growth conditions were determined. Western blot analysis revealed that V-ATPase of S. salsa consists of at least nine subunits (apparent molecular masses 66, 55, 52, 48, 36, 35, 29, 18, and 16 kDa). This polypeptide pattern did not depend on culture conditions. V-PPase is composed of a single polypeptide (69 kDa). An additional polypeptide (54 kDa) was detected in the fractions of NaCl-, KCl- and PEG-treated plants. It turned out that the main strategy of salt-tolerance of S. salsa seems to be an up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, while V-PPase plays only a minor role. The increase in V-ATPase activity is not obtained by structural changes of the enzyme, but by an increase in V-ATPase protein amount.     

Factors influencing the storability of Fagus sylvatica L. seeds after release from dormancy

Seeds of beech (Fagus sylvatica L.) that have been subjected to dormancy breaking consisting of 10 weeks of prechilling at 3 °C and 34 % water content (WC) and then desiccation to 10 % WC, are non-dormant (ND). ND seeds are characterised by greater sensitivity to storage conditions, than no prechilled, dormant (D) seeds. The aim of the present work was to investigate factors affecting the loss of seed viability during storage of D and ND beech seeds at different temperatures (4 and 20 °C) and humidity levels (45 and 75 % RH) for 3 weeks. In general, both D and ND seeds maintained a high germination capacity after storage at 4 °C. At 20 °C and 45 and 75 % RH the germination capacity of D seeds diminished to 80 and 28 %, respectively. Under the same conditions, ND seeds lost germination capacity to a greater degree, with only 62 and 7 % germinated seeds, respectively. At 20 °C, an increase in production of reactive oxygen species was observed, and the increase was significantly higher in ND seeds. The loss of germination capacity was coincident with an increase in electrolyte leakage and accumulation of free fatty acids, which suggests that membrane deterioration was the cause of the decline in germinability. ND seeds stored at 20 °C and 45 and 75 % RH showed a greater decrease than D seeds in contents of the primary phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as well as in polyunsaturated fatty acids (18:2 and 18:3). ND seeds possessed more unsaturated fatty acids, especially 18:3, than D seeds in the phospholipid fraction before storage. D seeds were characterised by a significantly higher level of α-tocopherol and UV-absorbing phenols. The level of ascorbate was similar in D and ND seeds. D seeds contained glutathione in both reduced (GSH) and oxidised (GSSG) forms, and GSSG dominated GSH. ND seeds contained more GSSG form than D seeds. We concluded that the membranes of ND seeds are exposed to greater oxidative stress during storage due to higher levels of unsaturation and lower levels of α-tocopherol, the main antioxidant that protects membranes against free radical attack.