Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.     

Macrophage supernatants have both stimulatory and suppressive effects on mesangial cell proliferation

Macrophages may modulate mesangial expansion following renal injury via secretory products. We undertook the present study to determine the effects of macrophages supernatants on mesangial cell proliferation. Macrophage supernatants collected in serum-free media after 24 hours caused significantly enhanced mesangial cell proliferation in long-term culture at concentration up to 50% but caused suppression at higher concentration (control, 122,000 ± 14,000 cells/well: 50% supernatant, 188,000 ± 15,100 cells/well, p < 0.02 compared to control, n = 4; 80% supernatant, 52,000 ± 3,500 cells/well, P < 0.01 compared to control, n = 4). In short-term culture [³H] thymidine incorporation, a measure of DNA synthesis, was significantly enhanced compared to control at supernatant concentrations up to 30% (30% supernatant, 4,120 ± 310 cpm/well; control, 3,210 ± 97 cpm/well, P < 0.5, n = 4), but uptake was reduced at high concentration (80% supernatant, 2,900 ± 74 cpm/well; control, 3,210 ± 97 cpm/well, P < 0.05, n= 4) When macrophages supernatants were collected after 48 hours incubation and incubated with mesangial cells, mesangial cell thymidine uptake was significantly suppressed compared to control (48-hours supernatant, 4,060 ± 260 cpm/well; control, 5,890 ± 270 cpm/well, P < 0.01, n = 4) and comapared to 24-hour supernatants, which enhanced uptake (24-hour supernatant, 8,080 ± 340 cpm/well; control, 5,890 ± 270 cpm/well, P < 0.01, n =4). Our results suggest that macrophages supernatants can directly enhance mesangial cell proliferation in vitro in both short-term and long-term culture, though this effect is lost at high concentrations of supernatant. These data lend support to the potential role of the macrophage in mediating mesangial expansion following renal injury. © 1993 Wiley-Liss, Inc.     

The rice coleoptile: an example of anaerobic nitrate assimilation

Nitrate present in rice caryopses can be reduced to ammonium and the ammonium subsequently assimilated by the coleoptile during anaerobic germination. All the enzymes of nitrate reduction and ammonia assimilation are present in the coleoptile. The supply of ¹⁵NO₃ confirms that the nitrate nitrogen is anaerobically incorporated into amino acids. Under anoxia, nitrate and nitrite reductase activities are increased in the coleoptile by exogenous nitrate. The importance of nitrate utilization during the anaerobic germination of rice caryopses is discussed.     

Abscisic acid induced stress-like polyamine pattern in wheat seedlings, and its reversal by potassium ions

In 3-day-old wheat ( Triticum aestivum L. cv. Marinat) seedlings, 100 μ M ABA blocked the growth and altered the level of K⁺ in both the shoot and root. The presence of ABA increased the putrescine titer during a 24-h treatment. Increasing the endogenous level of K⁺ by the addition of 10 m M KCl to the ABA-treated seedlings, inhibited the effect of ABA on growth and putrescine level. In both tissues, ABA increased putrescine content at low concentrations (1 μ M ), reaching the maximal effect at 100 μ M . Putrescine increase induced by ABA was inhibited by both α-difluoromethylarginine (DFMA) and α-difluoromethylornithine (DFMO) in shoots while only the inhibitor of arginine decarboxylase was effective in the root. The presence of ABA modulated, in opposite ways, ornithine and arginine decarboxylase activities. These results are discussed in relation to ion balance under stress.     

Oxidation of extracellular matrix modulates susceptibility to degradation by the mesangial matrix metalloproteinase-2.

Protein oxidation occurs in aging and in various inflammatory conditions. Glomerulosclerosis is characterized by an accumulation of extracellular matrix (ECM) proteins and a paucity of glomerular mesangial cells and can be seen as an end-result of glomerular injury and in aging. ECM accumulation is the net result of the balance between synthesis and degradation. ECM may become oxidized as a part of inflammatory renal injury and with aging. We evaluated the hypothesis that oxidation of mesangial ECM could alter its susceptibility to the action of ECM degrading enzymes. Radiolabeled mesangial ECM was generated by growing cells on tissue culture plastic and incubating with [3H]proline. After removal of cells, leaving behind ECM, selected wells were oxidized using a FeCl3/EDTA/ascorbate system or treated under control conditions. The control and oxidized matrices were then incubated with concentrated supernatants from mesangial cells containing the major mesangial ECM degrading enzyme, the matrix metalloproteinase-2, whose activity was confirmed by gelatin substrate zymography. Counts released corresponding with ECM degraded were measured. ECM oxidized with this system was significantly less susceptible to degradation compared to control ECM. To confirm that this effect was specifically due to oxidative modification of the ECM rather than changes unrelated to oxidation we coincubated ECM with the oxidizing system plus the radical spin trap N-tert-butyl-alpha-phenylnitrone (PBN). PBN treatment was able to prevent the impaired susceptibility to degradation induced by exposure to the oxidizing system. Exposure of ECM to milder oxidative stress, however, modestly enhanced susceptibility to degradation. These data suggest that oxidation of mesangial ECM can modulate its susceptibility to degradation. This may account for the development of ECM accumulation and glomerulosclerosis in inflammatory renal injury and in aging.     

Geographic isolation affects inter- and intra-specific seed variability in the Astragalus tragacantha complex,as assessed by morpho-colorimetric analysis

The effect of geographical isolation on the inter- and intra-specific seed variability of the Astragalus tragacantha complex was investigated by using a computer-aided imaging system. In particular, seed morphometric and colorimetric features of Astragalus balearicus, A. tegulensis, A. terraccianoi, A. thermensis and A. tragacantha were measured and the data were used to discriminate among regions of provenance, taxa and populations. Discriminant analysis of the data supports the accepted taxonomy of the group and the hypothesis that interspecific differentiation resulted from geographical isolation. The results also showed a positive correlation between the wideness of species’ distribution and the levels of seed intra-specific variability.     

Effects of NaCl stress on seed germination and seedling development of Brassica insularis Moris (Brassicaceae)

• Brassica insularis is a protected plant that grows on both coastal and inland cliffs in the western Mediterranean Basin. The objective of this study was to test if any variability exists in the salt stress response during seed germination and seedling development in this species relative to its provenance habitat.
• Variability among three populations in the salt stress effects on seed germination and recovery under different temperatures was evaluated. The effect of nebulisation of a salt solution on seedling development was evaluated between populations growing at different distances from the sea.
• Seeds of B. insularis could germinate at NaCl concentrations up to 200 mm . Seed viability was negatively affected by salt, and recovery ability decreased with increasing temperature or salinity. Inter‐population variability was detected in salt response during the seed germination phase, as well as in seedling salt spray tolerance. The inland population seedlings had drastically decreased survival and life span and failed to survive to the end of the experiment. In contrast, at least 90% of the coastal seedlings survived, even when sprayed at the highest frequency with salt solution.
• This study allowed investigation of two natural factors, soil salinity and marine aerosols, widely present in the B. insularis habitat, and provided the first insights into ecology of this protected species and its distribution in the Mediterranean. These results might be useful in understanding the actual distributions of other species with the same ecology that experience these same abiotic parameters.

     

The regulatory elements of araBAD operon,contrary to lac‐based expression systems,afford hypersynthesis of murine,and human interferons in Escherichia coli

The overexpression of four different interferons, i.e., murine interferon α1 and human interferons α1, α8, and α21 was challenged in Escherichia coli. Synthetic genes coding for these interferons were designed, assembled, and cloned into the vector pET9a (using the NdeI and BamHI sites), placing interferon expression under the control of phage T7 promoter. Despite an intensive screening for optimal culture conditions, no interferon synthesis was observed using overexpression systems based on the regulatory elements of lac operon (e.g., in E. coli BL21DE3). On the contrary, high levels of interferon expression were detected in E. coli BL21AI, which chromosome contains the gene coding for phage T7 RNA polymerase under the control of the araBAD promoter. To analyze the reasons of this striking difference, the molecular events associated with the lack of interferon expression in E. coli BL21DE3 were studied, and murine interferon α1 was chosen as a model system. Surprisingly, it was observed that this interferon represses the synthesis of T7 RNA polymerase in E. coli BL21DE3 and, in particular, the expression of lac operon. In fact, by determining β‐galactosidase activity in E. coli BL21AI, a significantly lower LacZ activity was observed in cells induced to interferon synthesis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Light,temperature, dry after‐ripening and salt stress effects on seed germination of Phleum sardoum (Hackel) Hackel

Phleum sardoum is an endemic psammophilous species of Sardinia, growing exclusively on coastal sandy dunes. The effect of glumes on seed germination, germination requirements at constant (5–25°C) and alternating (25/10°C) temperatures, both in the light (12/12 h) and in the dark were evaluated, as well as the effect of a dry after‐ripening period (90 days at 25°C), the salt stress effect (0–600 mmol NaCl) and its recovery on seed germination. The presence of glumes reduced final germination percentages. For fresh naked seeds, high germination percentages were observed at 10°C. Dry after‐ripening increased germination rate at low temperatures, but did not affect final germination percentages. NaCl determined a secondary salt‐induced dormancy which recovery interrupted only partially. Our results highlighted that this species has its optimum of germination during autumn–winter when, under a Mediterranean climate, water availability is highest and soil salinity levels are minimal.