Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: nitrogen fluxes within the plant and changes in soluble protein patterns

Oilseed rape (Brassica napus L.) is commonly grown for oil or bio-fuel production, while the seed residues can be used for animal feed. It can also be grown as a catch crop because of its efficiency in extracting mineral N from the soil profile. However, the N harvest index is usually low, due in part to a low ability to remobilize N from leaves and to the fall of N-rich leaves which allows a significant amount of N to return to the environment. In order to understand how N filling of pods occurs, experiments were undertaken to quantify N flows within the plant by (15)N labelling and to follow the changes in soluble protein profiles of tissues presumed to store and subsequently to remobilize N. Whereas N uptake increased as a function of growth, N uptake capacity decreased at flowering to a non-significant level during pod filling. However, large amounts of endogenous N were transferred from the leaves to the stems and to taproots which acted as a buffering storage compartment later used to supply the reproductive tissue. About 15% of the total N cycling through the plant were lost through leaf fall and 48%, nearly all of which had been remobilized from vegetative tissues, were finally recovered in the mature pods. SDS-PAGE analysis revealed that large amounts of a 23 kDa polypeptide accumulated in the taproots during flowering and was later fully hydrolysed. Its putative function of storage protein is further supported by the fact that when plants were grown at lower temperature, both flowering, its accumulation and further mobilization were delayed. The overall results are discussed in relation to plant strategies which optimize N cycling to reproductive sinks by means of buffering vegetative tissues such as stems and taproots.     

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.     

Bacterial DNA methylation and gene transfer efficiency

The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences. These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam(-), dcm(-)) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam(-), dcm(-)) counterpart. Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene transfer efficiency. However, the persistence of the use of (dam(+), dcm(+)) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in eukaryotic cells or organisms.     

Effects of a localized supply of nitrate on NO3 - uptake rate and growth of roots in Lolium multiflorum Lam.

Roots of higher plants are usually exposed to varying spatial and temporal changes in concentrations of soil mineral nitrogen. A split root system was used to see how Lolium multiflorum Lam. roots adapt to such variations to cope with their N requirements. Plants were grown in hydroponic culture with their root system split in two spatially separated compartments allowing them to be fed with or without KNO₃. Net NO₃ ^- uptake, ¹⁵NO₃ ^- influx and root growth were studied in relation to time. Within less than 24 h following deprivation of KNO₃ to half the roots, the influx in NO₃ ^- fed roots was observed to increase (about 200% of the influx measured in plant uniformly NO₃ ^- supplied control plant) thereby compensating the whole plant for the lack of uptake by the N deprived roots. Due to the large NO₃ ^- concentrations in the roots, the NO₃ ^- efflux was also increased so that the net uptake rate increased only slightly (35% maximum) compared with the values obtained for control plants uniformly supplied with NO₃ ^-. This increase in net NO₃ ^- uptake rate was not sufficient to compensate the deficit in N uptake rate of the NO₃ ^- deprived split root in the short term. Over a longer period (>1 wk), root growth of the part of the root system locally supplied with NO₃ ^- was stimulated. An increase in root growth was mainly responsable for the greater uptake of nitrate in Lolium multiflorum so that it was able to fully compensate the deficit in N uptake rate of the NO₃ ^- deprived split root.     

Both Hepatic and Body Iron Stores Are Increased in Dysmetabolic Iron Overload Syndrome. A Case-Control Study

Background & Aims

Hepatic iron is increased in dysmetabolic iron overload syndrome (DIOS). Whether this reflects elevated body iron stores is still debated. The study was aimed at assessing body iron stores in DIOS patients by calculating the amount of mobilized iron (AMI).

Methods

We conducted a prospective case-control study comparing AMI in 12 DIOS patients and 12 overweight normoferritinemic subjects matched on BMI and age. All participants were phlebotomized until serum ferritin dropped ≤ 50μg/L.

Results

The two groups were comparable with respect to metabolic abnormalities and differed according to serum ferritin levels only. AMI was significantly (p<0.0001) higher in DIOS (2.5g±0.7) than in controls (0.8g±0.3). No side effects were related to phlebotomies.     

Most significant genome regions involved in the control of earliness traits in bread wheat, as revealed by QTL meta-analysis

Earliness is one of the most important adaptation traits in plant breeding. Our purpose was to identify the genome regions of bread wheat involved in the control of earliness and its three components: photoperiod sensitivity (PS), vernalization requirement (VR) and intrinsic earliness (IE). A QTL meta-analysis was carried out to examine the replicability of QTL across 13 independent studies and to propose meta-QTL (MQTL). Initial QTL were projected on a recent consensus map (2004). Quality criteria were proposed to assess the reliability of this projection. These criteria were based on the distances between markers in the QTL regions. Chromosomes of groups 2 and 5 had a greater incidence on earliness control as they carry the known, major genes Ppd and Vrn. Other chromosome regions played an intermediate role in earliness control: 4A [heading date (HD) Meta-QTL], 4B (HD MQTL), 2B (VR MQTL) and 5B (IE MQTL). Markers at this four MQTL should prove helpful in marker-assisted selection, to better control earliness.     

8-Chloro-cyclic AMP and protein kinase A I-selective cyclic AMP analogs inhibit cancer cell growth through different mechanisms

Cyclic AMP (cAMP) inhibits the proliferation of several tumor cells. We previously reported an antiproliferative effect of PKA I-selective cAMP analogs (8-PIP-cAMP and 8-HA-cAMP) on two human cancer cell lines of different origin. 8-Cl-cAMP, another cAMP analog with known antiproliferative properties, has been investigated as a potential anticancer drug. Here, we compared the antiproliferative effect of 8-Cl-cAMP and the PKA I-selective cAMP analogs in three human cancer cell lines (ARO, NPA and WRO). 8-Cl-cAMP and the PKA I-selective cAMP analogs had similarly potent antiproliferative effects on the BRAF-positive ARO and NPA cells, but not on the BRAF-negative WRO cells, in which only 8-Cl-cAMP consistently inhibited cell growth. While treatment with the PKA I-selective cAMP analogs was associated with growth arrest, 8-Cl-cAMP induced apoptosis. To further investigate the actions of 8-Cl-cAMP and the PKA I-selective cAMP analogs, we analyzed their effects on signaling pathways involved in cell proliferation and apoptosis. Interestingly, the PKA I-selective cAMP analogs, but not 8-Cl-cAMP, inhibited ERK phosphorylation, whereas 8-Cl-cAMP alone induced a progressive phosphorylation of the p38 mitogen-activated protein kinase (MAPK), via activation of AMPK by its metabolite 8-Cl-adenosine. Importantly, the pro-apoptotic effect of 8-Cl-cAMP could be largely prevented by pharmacological inhibition of the p38 MAPK. Altogether, these data suggest that 8-Cl-cAMP and the PKA I-selective cAMP analogs, though of comparable antiproliferative potency, act through different mechanisms. PKA I-selective cAMP analogs induce growth arrest in cells carrying the BRAF oncogene, whereas 8-Cl-cAMP induce apoptosis, apparently through activation of the p38 MAPK pathway.